Autologous bone marrow stem cell transplantation in patients with liver failure: a meta-analytic review.

Stem Cells and Development Autologous bone marrow stem cell transplantation in patients with liver failure: a meta-analytic review (doi: 10.1089/scd.2014.0337) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof.

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1 TITLE: Autologous bone marrow stem cell transplantation in patients with liver failure: a meta-analytic review Running title: Stem cells and liver failure Author names: Kewei Wang, Xiaopan Chen*, Jinma Ren** Departments of Surgery, University of Illinois College of Medicine, Peoria, IL 61605, USA *Departments of Cancer Biology and Pharmacology, University of Illinois College of Medicine, Peoria, IL 61605, USA **Center for Outcomes Research/Department of Medicine, University of Illinois College of Medicine, Peoria, IL 61605, USA

Corresponding address: Kewei Wang, One Illini Drive, Peoria, IL 61605, USA. Phone/fax: 309-680-8617 Email: [email protected] Keywords: Liver failure, liver disease, bone marrow, stem cells, cytotherapy, meta-analysis Financial disclosures and/or conflicts of interest: The author has nothing to disclose. List of abbreviations: ABMSC, autologous bone marrow stem cells; HBV, hepatitis B virus; HCV, hepatitis C virus; PBC, primary biliary cirrhosis; PSC, primary sclerosing cholangitis; ATP, adenosine triphosphate; ALF, acute liver failure; ALT, alanine aminotransferase; AST, aspartate aminotransferase; ALB, albumin; TBIL, total bilirubin; PT, prothrombin time; PTA, prothrombin activity; INR, international normalized ratio; GCSF, granulocyte colony-stimulating factor; BUN, blood urea nitrogen; AFP, Alpha fetoprotein; HCC, hepatocellular carcinoma; MELD, model for end-stage liver disease; Keywords: Stem cell, bone marrow, transplantation, liver failure, meta-analysis

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2 ABSTRACT Autologous bone marrow stem cell (ABMSC) transplantation has been utilizing in clinical practice to treat patients with liver failure, but the therapeutic effect remains to be defined. A meta-analysis is essential to assess clinical advantages of ABMSC transplantation for patients with liver failure. A systematic search of published works (e.g. PubMed, Medline, Embase, Chin J Clinicians (Electronic edition) and Science Citation Index) was conducted to compare clinical outcomes of ABMSC transplantation in patients with liver failure. Meta-analytic results were tested by fixed-effects model or random-effects model, dependent on the characteristics of variables. A total of 534 patients from seven studies were included in final metaanalysis. Subsequent to ABMSC transplantation, there was no significant improvement in general symptom and sign such as loss of appetite, fatigue and ascites. Activities of serum ALT were not significantly decreased with weighted mean difference (WMD) of −19.36 and 95% confidence interval (CI) −57.53 to 18.80 (P = 0.32). Postoperative level of albumin (ALB) was expectedly enhanced by stem cell transplantation (WMD 2.97, 95% CI 0.52 to 5.43, P < 0.05, I2 = 84%). Coagulation function was improved as demonstrated by a short prothrombin time (PT) (WMD −1.18, 95% CI −2.32 to −0.03, P < 0.05, I2 = 6%), but was not reflected by prothrombin activity (PTA) (P = 0.39). Total bilirubin (TBIL) was drastically diminished after ABMSC therapy (WMD −14.85, 95% CI −20.39 to −9.32, P < 0.01, I2 = 73%). Model for end-stage liver disease (MELD) scores were dramatically reduced (WMD −2.27, 95% CI −3.53 to −1.02, P < 0.01, I2 = 0%). The advantage of ABMSC transplantation could be maintained more than 24 weeks as displayed by time-courses of ALB, TBIL and MELD score. ABMSC transplantation does provide beneficial effects for patients with liver failure. Therapeutic effects can last for six months. However, longterm effects need to be determined.

INTRODUCTION Liver failure is a life-threatening condition, in which the liver is losing or has lost all of its physiological function. The mortality rate highly reaches 40─80% [1]. Liver failure is a medical emergency and needs immediately intensive care. According to the difference of onset, the liver failure has three forms, acute,

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3 acute-on-chronic and chronic. Acute liver failure develops in a few days (as little as 48 hours) and can cause many complications such as infection, coagulopathy and encephalopathy. Acute-on-chronic liver failure can be seen in patients with previously diagnosed or undiagnosed chronic liver disease, manifesting as jaundice, coagulopathy, ascites and/or encephalopathy [2]. Most frequently, chronic liver failure advances gradually over a course of several months or years.

The liver failure is commonly caused by viral infection (HBV, HCV), alcoholism, drug overdose, cholestasis, poison (wild mushrooms, herbal medications), autoimmunity (PBC, PSC), inherited factor (hemochromatosis), or unknown origin. Causative factors induce liver injury via distinctive modes of cell death, including necrosis, necroptosis, autophagy and apoptosis [3]. The pre-existing genetic condition modifies susceptibility to various types of the causative factors and perpetuates the destruction of liver tissue [4]. These diverse modes of cell death consist in a dynamic spectrum during liver injury. The mode of liver cell death is doubtless determined by the duration and magnitude of etiological factors. When the extent of hepatocyte death is less than the capacity of liver regeneration, liver function can be compensated. Otherwise, liver failure occurs. The hepatocyte death contains two major types or apoptosis and necrosis [5]. Apoptosis as a temperate response is less severe than necrosis, but both modes of cell death often co-exist in liver disease, dependent on the severity of the insult. ATP as energy molecule is necessary for the execution of apoptosis, whereas ATP is depleted in necrotic tissue. Apoptosis is associated with little secondary impairment as compared with necrosis. The necrotic process recruits inflammatory cells (e.g. neutrophils) into liver parenchyma. Liver injury is further aggravated by inflammatory response, which leads to dramatically different outcome from apoptotic cell death in regard to progression of liver disease [6]. When the hepatic parenchyma is seriously damaged and poor function is unable to meet metabolic requirements of the body, a devastating syndrome of acute liver failure (ALF) develops. The crucial outcome is reflected by a series of clinical complications, such as coagulopathy, encephalopathy, hemodynamic changes, and electrolyte disturbance. There is a complicated relationship between liver injury and the syndrome of ALF. Current knowledge on ALF pathophysiology is limited due

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4 to the lack of an adequate animal model of the disease. Recent data indicate that necrosis is associated with poor outcome of ALF. The detection of necrosis can therefore become useful to identify ALF patients for liver transplantation. The liver failure can be diagnosed through a detailed history, sign and symptom, blood test, imaging test and liver biopsy. Early recognition of the ALF and an aggressive treatment can improve clinical outcome. Despite significant advance in medical care, however, the mortality remains extremely high.

Treatment of liver failure depends on information that clinicians promptly collect. For instance, an early detection of acute liver failure caused by an overdose of acetaminophen may decrease death rate. Likewise, if the liver failure is caused by viral infection, medical treatment and supportive care can alleviate symptoms of virus-induced liver injury, inhibit virus replication, and restore liver function. Intervention of liver failure includes (i) Medications to treat poisoning. Adequate medications can reverse effects of the toxins and relieve acute liver failure induced by acetaminophen overdose or mushroom poisoning; (ii) Treatment for complications. A positive treatment of complications can interrupt the deterioration of liver function and gives the liver a healing time. To improve symptoms and signs that patient is experiencing can impede disease progression, such as (a) relieving brain edema, (b) treating the infection and (c) preventing severe bleeding; (iii) Stem cell transplantation. For liver failure resulting from chronic liver disease, the guideline of initial treatment is to save still functioning part of the liver and to stimulate the regenerative capacity of the liver. A challenge is how to get enough hepatocytes for the recovery of liver function [7]. In recent years, a series of cytotherapeutics have been carrying out, such as autologous bone marrow mesenchymal stem cell transplantation, in vitro proliferation of bone marrow stem cells and transplantation, umbilical cord stem cell transplantation, and hepatocyte transplantation [8-11]. These therapies provide the best evidence for cell engraftment that promotes generation of functional liver parenchyma. In addition, ABMSC mobilized by an injection of granulocyte colonystimulating factor (GCSF), can also be included in the field of stem cell therapy [12]; (iv) Orthotopic liver transplantation. In many cases, acute liver failure is hard to be reversed. A liver transplant becomes

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5 necessary in clinical treatment. In fact, the liver transplantation is the most effective form for liver failure [13]. Sometimes, a liver transplant is the only cure for acute liver failure. Fortunately, the liver transplantation as a common procedure is often successful. Unfortunately, the liver transplantation is limited by rejection, adverse effects of immunosuppressive drugs, and especially a shortage of donor organ.

Animal experiments and clinical trials have shown beneficial effects of stem cell therapy [14, 15]. The intravenous injection of hematopoietic stem cells into mouse model of tyrosinemia type I could correct the metabolic defect and rescued the diseased animal. Another study also demonstrated that purified hematopoietic stem cells could differentiate into hepatocytes and stimulate the hepatic regeneration in vivo. Based on a set of animal data, some clinical studies are performed in patients with liver disease. Many prospective trials have evaluated the efficacy and safety of ABMSC transplantation. The ABMSC transplantation includes different cell types such as CD34, CD133, or unsorted cell mixture. Transfusion of cells can be via intrahepatic, intrasplenic or peripheral veins. The mechanism for transplantation of ABMSC to improve the situation of liver failure remains unknown. Potential therapeutic mechanisms involve regeneration, anti-apoptosis, inhibition of inflammation and regulation of immunity. Therapeutic effects of ABMSC transplantation can be estimated by the improvement of symptoms and signs, serum enzyme activities, biochemical markers (e.g. albumin, bilirubin, glucose and cholesterol), coagulation function, comprehensive MELD and Child-Pugh scores, mortality, survival rate and so on.

A significant advancement has been obtained as patients with liver failure were treated through the ABMSC transplantation [14-17]. However, the clinical outcomes are inconsistent. Moreover, an optimal method of the ABMSC transplantation for liver failure is still unknown. From laboratory and clinical perspective, the treatment of liver failure by the ABMSC transplantation is beneficial. Unfortunately, there is the heterogeneity in published studies. Some studies are based on small sample sizes with low statistical power, which cannot predict the result of a single large study. We therefore conducted a meta-

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6 analysis of ABMSC transplantation according to the standard of the Cochrane Collaboration. A metaanalysis based on systematic review of the literature can assess not only the effect and advantage of ABMSC transplantation in patients undergoing liver failure, but the strength of the evidence supporting such a favorable result as well. Results demonstrate the ABMSC transplantation can improve liver function in short term, but long-term effects need to be observed.

METHODS Systematical search of published work We systematically screened PubMed, Medline, Embase, Chin J Clinicians (Electronic edition) and Science Citation Index until April 2014. References were identified using the following keywords: “bone marrow stem cell” and “human liver disease”. The search of published work was not restricted by types of clinical studies. The relevant literature was further scrutinized by the completeness of data.

Study selection Trials eligible for inclusion were based on quality of evidence, included Grade I randomized controlled trials, Grade II-1 controlled trials without randomization and GradeII-2 cohort or case-control analytic studies [18]. We excluded studies with umbilicus cord stem cells and stem cells out of peripheral blood subsequent to a GCSF injection. Also, the exclusion covered studies that provided insufficient data relating to the pre-speciﬁed outcome variables. The included studies only contained patients with the transplantation of autologous bone marrow stem cells. Data extraction was accomplished by two investigators independently. Disagreement was resolved by third opinion.

Data collection and outcome measures We extracted general data on characteristics of participants (source of reference, country, study design, age, sex composition of patients), intervention and outcome measures in different studies. A primary comparison was performed among basic data out of ABMSC transplantation and control groups. We analyzed common symptoms and signs, levels of ALT, ALB, TBIL, PT/PTA/INR, MELD score, Child-Pugh

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7 score, morbidity and mortality. Morbidity was calculated by number of patients with at least one complication after ABMSC transplantation and mortality by in-hospital or at home death due to any cause. We subsequently excluded studies in which not enough data were reported. Meta-analysis was further performed including symptoms and signs, quantitative data of ALT, ALB, TBIL, PT/PTA, and MELD score.

Study quality assessment Bias in primary studies can lead to misleading summary estimates of accuracy. Quality assessment guides the interpretation of results in terms of potential for bias and sources of heterogeneity. The methodological quality of included studies was evaluated by random sequence generation, allocation concealment, blinding of participants and personnel, blinding of outcome assessors, incomplete outcome data, and selective reporting. Priori criteria of high quality study include (i) Randomized trials; (ii) Prospective cohort study; (iii) Consecutive patient enrollment; (iv) Adequately reported methodology of measurement.

Statistical analysis Review Manager (RevMan version 5.2, 2012, The Cochrane Collaboration, Oxford, UK) was used to pool data and meta-analysis. For categorical variable, treatment effect was expressed as odds ratio (OR) with corresponding 95% confidence intervals (CI). Results were compared through a random-effects model. For continuous variable, treatment effect was expressed as weighted mean difference (WMD) with corresponding 95% CI. Chi-square (Chi2 or χ2) and I2 statistics estimate the appropriateness of pooling individual study. Heterogeneity was evaluated by χ2-test with significance set at P-value 0.10. Heterogeneity was measured by I2 more than 50% as statistical significance. Forest plots were constructed with P-values of less than 0.05 as significant difference. RESULTS Quality assessment of the included studies

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8 A total of 1366 references were identified through the electronic search. After original full-texts had been reviewed, seven studies involving a total of 534 patients were included in present meta-analysis, which were from different countries Egypt, Brazil, Japan and China (Fig.1) [8, 19-24]. Resultant data were compared between stem cell transplantation (n = 291) with control (n = 243) groups. The risk of bias in the included studies was moderate (Fig. 2). The distribution of sample size and effect size could be outlined with a funnel plot. Owing to the small number of included studies, the funnel plot asymmetry was not investigated [25].

Description of the included studies A systematic review that covered stem cell treatment for human liver disease in recent years was summarized (Table 1). Following stem cell treatment, the liver function was improved at certain degrees as demonstrated in the different types of liver disease [8, 19-24, 26]. Here, types of stem cells include autologous bone marrow stem cells (ABMSC), in vitro proliferation of ABMSC, umbilical cord stem cells and GCSF-mobilized stem cells. Other parameters of stem cells were also compared, containing characteristics of stem cells, pattern of intervention, and clinical improvement subsequent to transplantation. Only studies using bone marrow ABMSC transplantation were chosen for further metaanalysis. General characteristics of the included studies were reflected by source of data, study design, number of patients, ratio of male/female, and age (Table 2). Of note, Peng study also randomized cirrhotic patients into stem cell transplantation group (73.58%) and control group (73.33%) (P = 0.973).

Improvement of clinical symptoms and signs Short-term therapeutic effects or outcomes subsequent to stem cell transplantation could be reflected by an improvement of clinical symptom and sign such as pain, nausea, loss of appetite, fatigue, dizziness, jaundice, edema, abdominal distension, ascites, pruritis, tremor, complications (bleeding, erythema, encephalopathy), and so on. According to an availability of data, only loss of appetite, fatigue and ascites were selected to represent patient’s changes in clinical symptom and sign. Studies by El-Ansary, Lyra,

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9 and Saito did not provide information on appetite and fatigue. Amer study gave a mean fatigue impact scale. Therefore, they were excluded in meta-analysis. A random-effect model was used to investigate the therapeutic effect of ABMSC transplantation when I2 ≥ 50%. There was no significant difference in loss of appetite between the stem cell transplantation and control groups (I2 = 50%, OR, 1.66; 95% CI, 0.90–3.07; P = 0.11) (Fig. 3a). Information on the number of patients in fatigue symptom was available for three studies relating to 429 patients (Fig. 3b). No improvement in fatigue symptom was found (I2 = 93%, OR, 2.62; 95% CI, 0.48–14.30; P = 0.27). Although individualized studies by El-Ansary and Sun showed ascites was significantly improved after stem cell infusion, there was no statistical difference in meta-analysis of ascites between ABMSC transplantation and control groups (I2 = 82%, OR, 2.09; 95% CI, 0.64–6.79; P = 0.22) (Fig. 3c).

Improvement of liver function by levels of ALT, ALB, TBIL, PT/PTA/INR Serum ALT activity is a basic marker to estimate liver injury (Table 3). Studies by Mao, Sun, and Peng et al provided primary data on ALT activity. El-Ansary study reported enzyme activities of ALT, ALP, GGT and AST as median (range). Studies by Lyra, Saito and Amer did not give any information on ALT activities. Meta-analysis of ALT activities indicated that the postoperative ALT level was not significantly altered between the transplantation and control groups (WMD, −19.36; 95% CI, −57.53 to 18.80; P = 0.32; I2 = 99%) (Fig. 4a). Time-course of ALT activity was provided by Peng et al from postoperative week 1 to week 48. The ALT levels showed significant decrease in the ABMSC transplantation group compared with the control group during 36 weeks. Albumin is an essential part of liver function panel (Table 4). Amer study presented serum albumin as figure. Lyra study showed albumin change in percentage and El-Ansary study provided albumin data as median (range). So they were not eligible for meta-analysis. Postoperative level of albumin was higher in the stem cell group than the control group (WMD, 2.97; 95% CI, 0.52–5.43; P < 0.05; I2 = 84%) (Fig. 4b). Time-course of ALB by Peng et al showed that ALB was quickly enhanced subsequent to ABMSC transplantation during 3-24 weeks. However, no significant difference was found after 48 weeks between ABMSC and control groups. For TBIL

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10 measurement, El-Ansary study reported TBIL level as median (range), Lyra study as figure, and Amer/Saito studies as a narrative description or “there was no difference between patients and control groups regarding serum bilirubin”. Other three trials provided available data on postoperative levels of bilirubin. Meta-analysis of the bilirubin showed a significant difference in the ABMSC transplantation group compared with control group (WMD, −14.85; 95% CI, −20.39 to −9.32; P < 0.01; I2 = 73%) (Fig. 4c). Time-course of TBIL by Peng et al revealed that serum TBIL were significantly decreased from postoperative week 4 to week 12, but no difference after 36 weeks between transplantation and control groups. The improvement of TBIL in stem cell group was markedly superior to those in control group during 4-12 week after ABMSC transplantation. Moreover, biochemical levels ALB, TBIL and ALT in Mao study were analyzed under Child–Pugh A&B and Child–Pugh C grades, respectively. Stem cell transplantation significantly improved biochemical levels in patients with Child–Pugh C grade. Coagulation function could be determined by PT, PTA or INR values. PT and PTA data were unavailable in Amer study, but a description indicated no INR difference between stem cell therapy and control groups. Lyra study reported the coagulation function as INR not PT/PTA. Meta-analysis of PT showed an improvement in stem cell group (WMD, −1.18; 95% CI, −2.32 to −0.03; P < 0.05; I2 = 6%), but PTA levels were unchanged between two groups (P = 0.39) (Fig. 4d, 4e). After stem cell transplantation, the improvement in PT scores of ABMSC group was significantly superior to those of control group during 412 week as demonstrated by Peng study.

Improvement of liver function can be indirectly reflected by changes of kidney function, glucose, total cholesterol, haemogram, and tumorigenic AFP expression Postoperative kidney function was monitored by levels of urine volume, creatinine and BUN in Sun study and the concentration of creatinine in El-Ansary study as median (range). However, not enough information from other studies was reported. Thus it was unable to perform meta-analysis of kidney function. Although levels of glucose and total cholesterol could also affected by liver disease, but relevant data were not available to run meta-analysis. Hemoglobin, leukocyte and platelet counts were presented

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11 in El-Ansary study as median (range). No haemogram information from other studies was provided. AFP, a tumorigenic marker of hepatocellular carcinoma (HCC), was measured only in El-Ansary study following stem cell transplantation. In Peng study, one case in stem cell group suffered from HCC at the 20th week after transplantation and nine cases in control group developed HCC throughout the 48-week follow-up (P = 0.107). Furthermore, there was no difference in incidence of HCC during 192 weeks of follow-up. The included studies did not provide enough information to do meta-analysis of carcinogenesis.

Therapeutic effects of ABMSC transplantation were evaluated by comprehensive MELD score, Child-Pugh score, liver volume, mortality and morbidity Single marker can estimate one aspect of liver function. Therapeutic effects of ABMSC transplantation need to be evaluated by comprehensive markers such as MELD score, Child-Pugh score, liver volume, mortality and morbidity. A meta-analysis of Amer and Peng studies disclosed a significant improvement of MELD score following ABMSC transplantation (WMD, −2.27; 95% CI, −3.53 to −1.02; P < 0.01; I2 = 0%) (Fig. 5). In Peng study, the improvement of MELD score in ABMSC group was prominently superior to that of control group during 3-36 weeks after transplantation. Child-Pugh scores were provided in ElAnsary study (in percentage). A time-course of Child-Pugh scores in Amer study showed a significant improvement during six month after stem cell transplantation. These results revealed that there were significantly low MELD and Child-Pugh scores subsequent to the stem cell transplantation. In Saito study, the functional liver volume in cm3 was obtained using single photon emission CT with a radiolabeled TcGSA and calculated by the outline extraction method. The functional index was improved during two weeks in 4 of the 5 patients who received stem cell therapy. In Sun study, one patient died in control group and one patient died in stem cell group respectively. In Peng study, no death number was available, but it reported no dramatic difference in mortality during 192 weeks of follow-up. Additionally, survival rate was plotted as figure in Peng study. No significant difference in survival rate was found

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12 between two groups. The meta-analysis of complications or morbidity in the included studies also demonstrated no statistical difference between stem cell transplantation and control groups.

DISCUSSION Major Finding The ABMSC transplantation has been clinically practicing in the treatment of liver failure. A meta-analysis including all of available data can provide a weighted average of effect size. Meta-analysis results of included studies demonstrate that the ABMSC transplantation does not alleviate the common symptom and sign as reflected by loss of appetite, fatigue and ascites. Serum levels of ALT activities were not significantly improved by ABMSC therapy. However, the ABMSC transplantation is truly beneficial to patients with liver failure in the improvement of coagulation function (PT), albumin (ALB), bilirubin metabolism (TBIL) and severity of liver disease (MELD). These favorable effects can last at least six months, but long-term effects remain to be determined.

Mechanisms of therapeutic ABMSC transplantation The improvement of liver function is found following ABMSC transplantation in short term. This finding supports the hypothesis that stem cells may be directly involved in the regeneration and repair of liver tissue. The exact mechanisms of stem cell therapy are unknown. Liver-protective effect of ABMSC transplantation may be via several aspects: (a) to stimulate liver regeneration. The regenerative response of liver injury mostly depends upon mature parenchymal cells. When native regenerative abilities of parenchymal cells have been exhausted, a foreign stimulation or supplementation becomes a necessity. Only rigorously purified hematopoietic stem cells can promote hepatocyte proliferation [14, 16, 26]; (b) to differentiate directly into parenchymal hepatocytes to compensate for the cell loss; (c) to secrete protective factors that prevent progressive apoptosis of functional cells and stimulate replication of host cells; (d) to regulate immunologic response of host liver; (e) preventing the liver fibrogenesis via a variety of cytokines, such as HGF, interleukin-6 and -10; (f) angiogenesis; and (g) to dissolve fibrosis directly

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13 through the production of matrix metalloproteinase [27-35]. Moreover, the ABMSC transplantation promotes cell fusion with parenchymal cells for liver regeneration [14, 16, 34, 36]. The cell fusion causes the concern for safety due to theoretical possible contribution to tumor formation. Notably, stem cells have profibrogenic potential and may contribute to scar-forming myofibroblasts in liver [37, 38]. Transplanted MSCs can migrate into liver parenchyma to stimulate cell differentiation, particularly under conditions of chronic injury. Previous studies revealed that hepatocyte-like cells were limited to a low number; by contrast, myofibroblast-like cells were observed in a significant number. Thus, profibrogenic potential of MSCs has to be noticed. Because of multiple functions of ABMSC, it is no surprise that the ABMSC transplantation can improve the prognosis of liver disease. Meta-analysis of available studies demonstrated that there was significant improvement of relevant biomarkers subsequent to ABMSC transplantation, which again confirms the mechanism and advantage of stem cell therapy in the recovery phase of liver injury.

Clinical significance The stem cell therapy provides new strategy for the treatment of both primary and secondary liver injury. The ABMSC transplantation has much beneficial role in the improvement of liver failure [8, 19, 21]. Therefore, its clinical application becomes popular in recent years. The following observations are noticed: (i) Stem cell transplantation could be through different routes such as intrasplenic, intrahepatic and peripheral vein. In the included studies, only one intrasplenic research was performed by Amer team. No statistical difference was observed between intrahepatic and intrasplenic groups. Both hepatic and splenic groups had a consistent outcome as regards jaundice, edema, serum albumin, serum bilirubin, liver enzymes, hemoglobin, total leucocytic count, or platelet; (ii) Intrahepatic route was again subdivided into hepatic artery and portal vein pathways. Sun study demonstrated a transplantation of stem cells via either hepatic artery (87 patients) or portal vein (64 patients) resulted in a similar improvement in levels of ALT, ALB, TBIL, PTA, urine volume, BUN, and creatinine. An injection through portal vein was better than via hepatic artery, because of relatively low percentage in adverse response and complications (e.g.

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14 fever, fatigue, nausea, dizziness, bleeding, hepatalgia, hematoma, low leucocytic count, and thrombosis) via portal vein transplantation. Indeed, in contrast to control group, the liver failure can be ameliorated with appropriate stem cell therapy. Remarkably, the benefit of ABMSC transplantation is based on the small number of included studies and mainly time-course data from Peng study. The additional evidence is thus needed to support the anticipated conclusion. Advantages of ABMSC transplantation can be reflected by the improvement of clinical symptoms and signs, biochemical levels of liver function, MELD score, Child-Pugh score, morbidity and mortality [8, 19, 20]. Other medical therapies such as antivirus, the treatment of complications and supportive therapeutics can be combined with ABMSC transplantation against liver failure. An increased possibility of tumorigenesis in stem cell-treated patients further highlights the need for a regular evaluation of cancerous risk [39, 40]. Potential mechanisms that lead to tumorigenesis subsequent to stem cell treatment implicate transdifferentiation of bone marrow stem cells and cell fusion between pre-malignant cell and bone marrow stem cell. Comprehensively, the mechanisms of tumorigenesis are complicated and beyond the scope of this review.

Study Limitations This study has a few limitations. The number of patients included in this meta-analysis is small and the published work search may have not covered all relevant references. In the present meta-analysis, it was impossible to adjust or stratify for potential confounders. Age, an important confounding factor for liver failure, is associated with severity of clinical complications and functional activity of stem cells [41-43]. Although the included studies enrolled different ages of patients, we were unable to determine the effect of age on the strength of the association. Moreover, presence and extent of liver cirrhosis have an adverse impact on short-term prognosis of patients with liver failure. A meta-analysis has inherent weaknesses because of combining heterogeneous data sets. Current meta-analysis includes studies that they were of variable quality and provided insufficient information on potential sources of bias. Obvious heterogeneity had been observed among studies, because it was hard to match patient characteristics in all studies. A random-effects model was applied to examine variation between studies, but this might not

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15 rule out the effect of heterogeneity between studies. Furthermore, almost no information on therapeutic medications or supportive treatment was provided in the included studies. The potential impact of liverprotective therapy rather than stem cell transplantation could not be evaluated in this meta-analysis.

Perspective At present, some small sample studies have shown beneficial roles of ABMSC transplantation in an improvement of the liver failure, whereas conflicting results have been reported in other studies as well [44-46]. After having pooled data of available studies, we are able to re-explore the association between ABMSC transplantation and improvement of the liver failure. The estimated ORs and WMDs for these variables may be less robust than those of established individual study. However, these biomarkers are very common and are well known for their roles in liver function. Future challenges include (i) long-term, large sample size, and randomized controlled study. Several critical issues deserve immediate attention, such as the optimal type of transfused stem cells, the most effective number of stem cells, the best route of administration and the optimal therapeutic timing. It needs a long time to get answers for these issues. Clinical safety and long-term advantages of stem cell therapy should be also reinforced by a large-sized randomized controlled trial; (ii) Liver-protective mechanisms of stem cell transplantation. It is important to track the fate of the transfused stem cells in vivo. The transfused stem cells can stimulate liver regeneration and improve liver function. However, stem cells are profibrogenic as well. The relationship between transfused stem cells and the hepatic inflammatory/fibrotic microenvironments is still to be determined [47, 48]; (iii) to test different types of stem cells, e.g. umbilicus stem cells and adipose-derived stem cells; and (iv) reinfusion to improve long term benefits [20]. The reinfusion of adipose-derived adult stem cells has been utilizing to treat complex perianal fistulas [49, 50]. As a novel idea, the reinfusion of stem cells at regular intervals may be suitable for liver disease. If these challenges can be figured out, the clinical application of stem cell-based therapy will be warranted for the treatment of patients with liver disease.

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16 Summary Meta-analysis shows significant advantages of the ABMSC transplantation as demonstrated by the improvement of ALB, PT, TBIL and MELD score. Further evidence from animal experiments and controlled clinical trials is still needed to estimate the beneficial and adverse effects. Future prospective study should be designed to assess the optimal technique of stem cell therapy, to investigate long-term therapeutic effects and to clarify the underlying mechanisms. In conclusion, the liver function is significantly improved by the ABMSC transplantation in patients with liver failure for short term. This finding strengthens the hypothesis that stem cells play essential roles in the recovery phase of liver disease. The long-term effect of stem cell therapy is still to be determined. DISCLOSURE There are no commercial relationships or conflict of interest in connection with the submitted manuscript.

ACKNOWLEDGMENTS We appreciated Li Zhang’s help during data extraction and the preparation of manuscript.

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FIGURE LEGENDS

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Stem Cells and Development Autologous bone marrow stem cell transplantation in patients with liver failure: a meta-analytic review (doi: 10.1089/scd.2014.0337) This article has been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ from this proof. Page 19 of 28

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Figure 1. Flow chart of references that was identified and included respectively.

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Figure 2. Summary of potential bias in the identified trials.

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Figure 3 Forest plots illustrate outcomes of clinical symptom and sign in patients undergoing stem cell transplantation. (a) loss of appetite, (b) fatigue, (c) ascites. M-H, Mantel–Haenszel test; CI, confidence interval.

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Figure 4 Forest plots show an improvement of liver function as reflected by (a) ALT, (b) ALB, (c)TBIL, (d) PT, and (e)PTA. IV, inverse variance; CI, confidence interval; SD, standard deviation.

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Figure 5 Meta-analysis of MELD score in patients with the ABMSC transplantation. IV, inverse variance; CI, confidence interval; SD, standard deviation.

24

Stem Cells and Development Autologous bone marrow stem cell transplantation in patients with liver failure: a meta-analytic review (doi: 10.1089/scd.2014.0337) as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro

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25 Reference

Types of stem cells No. of patients

Site injection

Types of liver disease

Evidence of efficacy

Amin MA, 2013

BM-MSC

20 treatment

Intrahepatic

HCV induced liver cirrhosis

Improvement in ALB, TBIL, PT/INR, ALT and AST

Mao, 2013

BM-MSC

32 treatment, 32 control

Hepatic artery

HBV

Improvement in ALB, TBIL, and PT

Park, 2013

BM-CD34 depleted MSC

5 treatment

Hepatic artery

HBV, HCV, toxic and Wilson

Improvement in ALB, TBIL, INR, ALT, and CP

Am Esch JS, 2012

CD133+ cells


Portal vein

Liver malignancy

Improvement in daily liver growth, INR, and survival rate

El-Ansary, 2012

BM-MSC


Peripheral vein

HCV induced liver cirrhosis

Improvement in ALB, TBIL, PT and MELD

Garg V, 2012

GCSF mobilized CD34+ cells


G-CSF subcutaneously

Acute-on-chronic liver failure

Increased survival, reduced MELD scores in two months

Han HS, 2014

MNCs or CD34+ cells

1 MNCs, 2 CD34+, 1 control

Portal vein

Hepatocellular carcinoma

Improvements in liver volume, liver function, clinical score and ICG-R15

Shi M, 2012

Umbilical cord-MSC


Peripheral vein

HBV induced liver failure

Improved ALB, TBIL, ALT, cholinesterase, PT, MELD and survival rates

Sun, 2012

BM-MSC


Hepatic artery, portal vein

HBV, HCV, alcohol, PBC, AIH

Improvement in ALB, TBIL, PTA, ALT, Cr, and BUN

Zhang Z, 2012

Umbilical cord-MSC


Peripheral vein

HBV induced liver cirrhosis

Improved liver function, MELD and reduced ascites

Amer, 2011

BM-derived MSC


Intrasplenic, intrahepatic

HCV

Improved ascites and MELD

Peng, 2011

BM-MSC


Hepatic artery

HBV

Improved ALB, TBIL and MELD

Saito T, 2011

BM-MSC


Peripheral vein

Alcoholic

Improved ALB, PT and CP score

Kim JK, 2010

BM-MSC

10 treatment

Peripheral vein

HBV

Increased ALB, liver volume and CP score

Lyra, 2010

BM-MSC


Hepatic artery

HBV, HCV, alcoholic, cryptogenic

Improvement in ALB, TBIL, INR, CP and MELD

Kharaziha P, 2009

BM-MSC

8 treatment

Peripheral, portal vein

HBV, HCV, alcoholic, cryptogenic

Improved MELD and liver function

Han Y, 2008

G-CSF mobilized PBMCs



HBV induced liver cirrhosis

Improved ALB and CP score

Khan, 2008


4 treatment

Hepatic artery

HBV, HCV

Improvements in ALB, TBIL and ALT

Pai M, 2008

Cultured CD34+ cells

9 treatment

Hepatic artery

Alcoholic

Improved ALB, TBIL, liver enzyme and CP score

Spahr, 2008




Alcoholic

Increased serum HGF and proliferation of hepatic progenitor cells

Lyra AC, 2007

BM-MSC

10 treatment

Hepatic artery

HCV, alcoholic, cryptogenic

Improved TBIL, ALB, INR and CP

Furst, 2007

CD133+ cells


Portal vein

Chronic liver failure and cirrhosis

Increase in daily liver growth

Gasbarrini a, 2007


1 treatment

Portal vein

Drug-induced acute liver failure

Improved PT, AST, and ALT

MoHamadnejad M, 2007

BM-MSC

3 cryptogenic, 1 AIH

Peripheral vein

Cryptogenic, AIH

Improved MELD in 2 patients

MoHamadnejad M, 2007

CD34+ cells

4 treatment

Hepatic artery

HBV, AIH, PBC, cryptogenic

Improved MELDand albumin (2)

Yan, 2007


2 treatment

Hepatic artery

HBV

Improvement in ALB, TBIL, AST, ALT, and CP

Gordon MY, 2006

CD34+ cells

5 treatment

Portal vein, Hepatic artery

HBV, HCV, alcoholic, PSC

Improvement in albumin or bilirubin in 3/5 patients

Terai S, 2006

BM-MSC

5 HCV, 3 HBV, 1 unknown

Peripheral vein

HBV, HCV, cryptogenic

Improved ALB and CP

Yannaki 2006


2 treatment

Peripheral vein

Alcoholic

Improvement in the Child-Pugh and MELD Score

Am Esch JS, 2005

CD133+ cells


Portal vein

Liver cancer, no cirrhosis

2.5-fold increase in left lobe in study group on CT volumetry

Note: BM, bone marrow; MSC, mesenchymal stem cell; CP, Child-Pugh score; MELD, model for end- stage liver disease; AIH, autoimmune hepatitis;PB, peripheral blood; G-CSF, granulocyte colony-stimulating factor; INR, international normalized ratio; MNCs, mononuclear cells; ICG-R15, indocyanine green R15.

Table 1 A systematic review about stem cell therapy for liver disease

25

Stem Cells and Development Autologous bone marrow stem cell transplantation in patients with liver failure: a meta-analytic review (doi: 10.1089/scd.2014.0337) as been peer-reviewed and accepted for publication, but has yet to undergo copyediting and proof correction. The final published version may differ fro

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Table 2 General characteristics of studies included in the meta-analysis

Table 3 A comparison of ALT activity between ABMSC transplantation and control groups Source Mao, 2013 El-Ansary, 2012 Sun, 2012 Amer, 2011 Peng, 2011 Saito, 2011 Lyra, 2010

Country

Study Design

China Egypt China Egypt China Japan Brazil

Grade II-1 Grade I Grade II-1 Grade I Grade I Grade I Grade I

Age (subject; control)

Male : Female (subject; control)

56.2 ± 11.7; 53.5 ± 8.0 48.0 ± 7.4; 51.6 ± 7.2 Median: 49.5 (26 ~ 73) 50.5 ± 4.1; 50.0 ± 3.6 42.2 ± 10.8; 42.2 ±11.4 64.6 ± 2.9; 61.2 ± 4.5 56.7 ± 9.2; 50.0 ± 10.4

20:12; 26:6 11:4; 8:2 48:16; 16:4; 17:3 50:3; 99:6 5 : 0; 5 : 0 N/A

Note: (1) Grade I: randomized controlled trials; Grade II-1: controlled trials without randomization. (2) N/A: not available.

Source Mao, 2013 El-Ansary, 2012 Sun, 2012 Amer, 2011 Peng, 2011 Saito, 2011 Lyra, 2010

Pre-ABMSC 95.82± 66.61 42.0 (19.0–104.0) 79.53 ± 17.63 N/A 97.51 6± 56.60 N/A N/A

Control 96.33± 62.44 44.0 (17.0–66.0) 93.66 ± 17.89 N/A 98.26 6± 56.73 N/A N/A

P value

ABMSC

N.S. N/A N.S. N/A 0.97 N/A N/A

56.65 ± 9.56 54.0 (21.0–132.0) 34.85 ± 13.42 N/A 55.49 ± 25.91 N/A N/A

Control 58.40 ± 9.06 57.5 (25.0–70.0) 89.56 ± 17.54 N/A 56.89 ± 32.22 N/A N/A

P value N.S. 0.94

Cartilage Repair With Autologous Bone Marrow Mesenchymal Stem Cell Transplantation: Review of Preclinical and Clinical Studies.

Phase 1 Trial of Autologous Bone Marrow Stem Cell Transplantation in Patients with Spinal Cord Injury.

Angiogenesis induced by autologous whole bone marrow stem cells transplantation.

Autologous bone marrow transplantation in decompensated liver: Systematic review and meta-analysis.

Cytogenetics in autologous bone marrow transplantation.

Autologous bone marrow transplantation in solid tumors.

Autologous bone marrow transplantation in solid tumors.

Mesenchymal Bone Marrow-derived Stem Cells Transplantation in Patients with HCV Related Liver Cirrhosis.

Bone marrow plasma cell assessment before peripheral blood stem cell mobilization in patients with multiple myeloma undergoing autologous stem cell transplantation.

Autologous bone marrow transplantation for Hodgkin's disease.

Bronchiolitis obliterans after autologous bone marrow transplantation.

Effect of bone marrow mesenchymal stem cell transplantation on acute hepatic failure in rats.

Allogeneic bone marrow mesenchymal stem cell transplantation in patients with UDCA-resistant primary biliary cirrhosis.

Allogeneic hematopoietic stem cell transplantation for inherited bone marrow failure syndromes.

Late donor bone marrow failure after allogeneic hematopoietic stem cell transplantation.

Bone marrow transplantation in patients with thalassemia.

Allogeneic bone marrow mesenchymal stem cell transplantation for periodontal regeneration.

Bone Marrow GvHD after Allogeneic Hematopoietic Stem Cell Transplantation.

Immunologic purging of marrow assessed by PCR before autologous bone marrow transplantation for B-cell lymphoma.

Pulmonary complications in lymphoma patients treated with high-dose therapy autologous bone marrow transplantation.

Feasibility and safety of intrathecal transplantation of autologous bone marrow mesenchymal stem cells in horses.

Autologous Hematopoietic Stem Cell Transplantation in Dialysis-Dependent Myeloma Patients.

Autologous bone marrow mononuclear cells intrathecal transplantation in chronic stroke.

Autologous bone-marrow transplantation in relapsed adult acute leukaemia.