Nan Li1, Xueming Wang2, Yuling Shi1, Na Li3, Junshan Zhai1, Xin Wang1, Li’na Sha1, Chaohui Zhu3, Lin Zhang3, Yanmei Wang1, Kai Wang

1Department of Digestion, the 309 Hospital of Chinese PLA, Beijing 100091, China

2Department of Pharmacy, the 309 Hospital of Chinese PLA, Beijing 100091, China

3Department of Minimally Invasive Intervention, the 309 Hospital of Chinese PLA, Beijing 100091, China

Supported by a grant from Wu Jieping Medical Foundation, No.320.6710.10001*

Received: 2011-09-08
Accepted: 2011-10-10 

Li N, Wang XM, Shi YL, Li N, Zhai JS, Wang X, Sha LN, Zhu CH, Zhang L, Wang YM, Wang K. Transplantation of peripheral blood stem cells for treatment of brain injuries in cases with liver cirrhosis. Neural Regen Res.

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Abstract
BACKGROUND AND AIM: Although transplantation of bone marrow-derived stem cells has classically been used for treatment of liver cirrhosis and other diseases, it has certain disadvantages. The present study investigated the efficacy and safety of peripheral blood stem cell transplantation and dynamically monitored serum levels of alpha-fetoprotein and its variant alpha-fetoprotein-L3, a tumor marker, to investigate the relationship between transplantation efficacy and a malignant phenotype of liver cancer.
METHODS: Forty-four patients with decompensated liver cirrhosis underwent intrahepatic transplantation of autologous peripheral blood stem cells via the portal vein. At 3 to 5 months postoperatively, improvements in clinical symptoms were observed, and changes in prothrombin time and serum alanine transaminase, total bilirubin, albumin, alpha-fetoprotein, and alpha-fetoprotein-L3 levels were evaluated.
RESULTS: At 2 weeks postoperatively, prothrombin time and alanine transaminase, total bilirubin, and albumin levels began to recover toward normal levels. After 2 to 3 months postoperatively, greater changes in alanine transaminase, total bilirubin, and prothrombin time were found, while albumin did not recover until 6 months after surgery. There was no significant difference between the 2- and 6-month postoperative alpha-fetoprotein-L3 levels, and the positive rate of serum alpha-fetoprotein-L3 was not closely related to the alpha-fetoprotein level.
CONCLUSIONS: Transplantation of peripheral blood stem cells via the portal vein for treatment of decompensated liver cirrhosis was acceptable for its excellent efficacy, low costs, and low technical risk. There were no obvious changes in alpha-fetoprotein-L3 levels in patients with decompensated liver cirrhosis before or after surgery.
Key Words: stem cells, liver cirrhosis, alpha-fetoprotein


INTRODUCTION
   
In China, advanced liver cirrhosis is a disease with high morbidity and mortality, with a lack of practical and effective therapeutic methods. Several studies have demonstrated bone marrow-derived stem cell differentiation into hepatocytes in vivo and in vitro, suggesting that this widely used stem cell transplantation technique provides new options for the treatment of decompensated liver cirrhosis[1]. However, this method is associated with several problems[2]. A limited amount of marrow and blood mixture can be collected, which limits the number of stem cells obtained. This method also requires anesthesia, which may present problems depending on the patient’s clinical condition. Furthermore, there is the ethical issue of whether stem cell transplantation is associated with the occurrence of liver cancer.
Between October 2007 and February 2009, 44 patients with decompensated liver cirrhosis underwent intrahepatic transplantation of autologous peripheral blood stem cells (PBSCs) via the portal vein. Liver function parameters and clinical symptoms of the patients were examined after surgery. A series of biochemical indices and the levels of the tumor markers alpha-fetoprotein (AFP) and AFP-L3[3-4], an AFP variant, were measured in all patients. Liver function and clinical symptoms of patients with decompensated liver cirrhosis recovered to a certain degree after PBSC transplantation, and the AFP-L3 level did not indicate the presence of a malignant biological phenotype within the follow-up time period.

METHODS

Patients
A total of 44 inpatients with decompensated liver cirrhosis were clinically diagnosed by our hospital and included in this study. These patients were 25 males and 19 females (42.4 ± 11.9 years of age). Decompensated liver cirrhosis was caused by hepatitis B virus infection in 26 patients, hepatitis C virus infection in 14, and other factors in 4. All patients had class C Child-Pugh liver disease, and all met the diagnostic criteria for liver cirrhosis according to the 2000 Guidelines for the Prevention and Treatment of Viral Hepatitis[5]. Intrahepatic space-occupying diseases and hepatocellular carcinoma were excluded in all patients by type B ultrasonography, computed tomography, or magnetic resonance imaging.
Mobilization, collection, and isolation of PBSCs
Following a previously described protocol[6-7], granulocyte colony-stimulating factor (Kirin Kunpeng Bio-Pharmaceutical Co., Ltd., Japan) was subcutaneously administered to all patients at 300 μg/d for 2 to 3 successive days to mobilize PBSCs and monitor changes in PBSC numbers. During this period, patients were monitored for complications including osteodynia, hypodynamia, erythema, gastrointestinal tract reaction, spontaneous splenic rupture, severe pyogenic infection, and hypercoagulable state. When peripheral white blood cells reached 1×104/ml, 70 to 102 mL of stem cells were isolated during a single extraction using a blood cell separator (CS-3000 Plus, Baxter, Deerfield, IL). During this period, some adverse events were observed, including hypocalcemia-induced perioral numbness, convulsion, vasovagal reaction–induced pale face, hypovolemia-induced pale face, and loss of consciousness.
Cell morphology and quantification
The morphology of isolated PBSCs was observed using inverted phase contrast microscopy. Surviving cells were identified by trypan blue staining. Stem cells (70–102 mL) were isolated using a CS 3000 Plus Blood Cell Separator (Baxter, Deerfield, IL, USA). Then, the number of cells in 0.5 mL was counted by trypan blue staining and cell activity was observed through the use of an inverted phase contrast microscope.
Cell categorization by flow cytometry
The digested mononuclear cell suspension was centrifuged for 10 minutes, and the supernatant was discarded. The resultant product was washed with PBS twice, fixed with 4°C 70% ethanol, washed with PBS twice, resuspended with 50 μg/mL propidium iodide staining solution containing 50 μg/mL RNase, and incubated for 30 minutes at 37°C in the dark. The percentages of CD34+, CD1+, and CD2+ cells were calculated. In addition, blood smears were made for differential counts.
Cell transplantation pathway and method
After isolation, PBSCs were preserved at room temperature and transplanted within 4 hours. Following a previously described method[8], the right branch of the portal vein was subcutaneously and transhepatically punctured under X-ray guidance, and a catheter end was placed at the main portal vein for visualization. The PBSC suspension was slowly perfused via the main portal vein for 20 to 30 minutes.
Measurement of biochemical indices
The procedure of Heino and Hentunen was followed[8]. Preoperatively (morning sample following an overnight fast) and 3 days, 2 weeks, 1 month, 2 months, and 3 months postoperatively, venous blood samples were taken for measurements of prothrombin time (PT) and serum alanine transaminase (ALT), total bilirubin (TB), and albumin (ALB) levels using a biochemical analyzer (Beckman Instruments, Inc., Brea, CA). Preoperatively and 2 and 6 months postoperatively, 5 mL of venous blood was taken from each patient, centrifuged at 3000 rpm for serum separation, transferred into tubes, and preserved at -20°C for measurement of AFP and AFP-L3 levels. In accordance with the instructions of the AFP-L3 detection reagent kit (Beijing Biotechnology Co., Ltd., Beijing, China), serum levels of AFP and AFP-L3 were determined by chemiluminescent assay using an automated analyzer (cobas e 601; Roche Diagnostics, Indianapolis, IN). The ratio of the L3 fraction to total AFP (AFP-L3%) was also calculated. An AFP-L3% of ≥10% was considered to be a positive criterion of hepatocellular carcinoma.
Postoperative observation
Preoperatively and 3 days, 1 week, 1 month, 2 months, and 3 months postoperatively, changes in PT and serum ALT, TB, and ALB levels were determined. In addition, reductions in adverse events and improvements in clinical symptoms (e.g., hypodynamia, abdominal distention) were observed.
Statistical analysis
The data were statistically processed using SPSS 13.0 software (SPSS, Chicago, IL) and were expressed as mean ± standard deviation (SD). A t test was used for comparisons between levels prior to and after surgery for the same patient. A P value of <0.05 was considered statistically significant.

RESULTS

Cell morphology and quantification
Inverted phase contrast microscopy revealed that the majority of suspended cells were red blood cells, and trypan blue–stained cells were stem cells. The cell survival rate of >50 to 60 cells per high-power visual field met the requirement for back-transfusion of cells (Figure 1). Quantification of PBSCs yielded 1.02 × 105/kg to 2.79 × 106/kg.
Cell categorization by flow cytometry
CD34 is a marker of bone marrow- and blood-derived stem cells, and the CD34 positivity rate is generally used for the quantification of blood-derived positive stem cells. Flow cytometry results demonstrated that the proportion of CD34+ cells among all surviving PBSCs was 0.8% to 1.45% (Fig. 2 and Table 4), which meets the quantification requirement for stem cell efficacy.


Determination of liver function
Changes in liver function–related indices after PBSC transplantation are shown in Table 1. Serum levels of ALT and TB were slightly increased at 3 days postoperatively and recovered to preoperative levels after 1 week. At 2 weeks, 1 month, and 2 months postoperatively, each liver function–related index recovered toward normal levels. The progress of 11 patients was followed for 15 to 27 months (follow-up was discontinued in the remaining patients or their follow-up data were lost). Serum levels of TB gradually decreased from 82.31 ± 44.86 μmol/L preoperatively to 42.23 ± 33.74 μmol/L postoperatively in 9/11 (81.82%) patients. Serum levels of ALB gradually increased from 26.41 ± 2.75 g/L preoperatively to 32.68 ± 3.04 g/L postoperatively in 8/11 (72.73%) patients. PT activity gradually increased from 30.18% ± 10.3% preoperatively to 44.84% ± 21.1% postoperatively in 10/11 (90.91%) patients. Postoperative ALT, TB, and PT values in all 44 patients were significantly decreased compared with preoperative levels (P < 0.05). These indices were approaching normal levels 3 months postoperatively (P < 0.01), while the ALB level was significantly increased 3 months postoperatively (P < 0.05). Five patients experienced recovery of liver function 3 months postoperatively.

Detection of AFP and AFP-L3 levels
AFP and AFP-L3 levels are shown in Table 2. At 2 months postoperatively, AFP serum levels were significantly higher than preoperative and 6-month postoperative levels (P < 0.01). There was no significant difference between preoperative and postoperative AFP-L3% serum levels (P > 0.05).
Determination of AFP-L3% within different concentrations of AFP
There was no significant difference in AFP-L3% among different concentrations of AFP (P > 0.05), and no significant difference in AFP-L3 level existed among patients with different concentrations of AFP (P > 0.05) (Table 3).

Postoperative improvement in clinical symptoms
Within 3 months of PBSC transplantation, ascitic fluid was reduced in 63.64% (17/37) of patients, appetite improved in 72.73% (28/37), physical strength regained in 63.64% (37/37), and abdominal distension alleviated in 63.64% (18/37). Clinical symptoms, such as deepening jaundice, worsened in 2 patients, and 1 patient died of massive gastrointestinal hemorrhaging secondary to portal hypertension.

Adverse events of PBSC transplantation
Collection, isolation, and intrahepatic transplantation of PBSCs were successful in all 44 patients. Intraoperative and postoperative nausea were observed in 3 patients and postoperative fever in 2, while decreases in the number of peripheral blood white cells was not found in any patient. Ecchymoses secondary to hemorrhage at the venous puncture site of the forearm were observed in 3 patients and improved after local compression hemostasis and symptomatic treatment. With the exception of slight osteodynia in 1 patient, no adverse events or complications associated with mobilization, collection, isolation, or transplantation of PBSCs were observed, such as skin rash, fever, muscular, spasm, or hemorrhage.

DISCUSSION

Several clinical reports have described transplantation of autologous stem cells for treatment of liver cirrhosis. However, issues concerning prognosis and improvement of liver cirrhosis following stem cell transplantation, stem cell differentiation in the injured liver, and malignant phenotypes have arisen[10,11]. Bone marrow stromal cells (MSCs) are primarily harvested by bone marrow puncture for treatment of liver cirrhosis and other diseases[5-7]. Transplantation of bone marrow-derived stem cells is a classic transplantation method with reliably curative effects. A drawback of this method is that just 80 to 120 mL of a bone marrow and blood mixture is collected to harvest the stem cells, limiting the number of stem cells and mixing them with peripheral blood. Therefore, the amount obtained is insufficient to overcome the hypersplenism associated with decompensated liver cirrhosis. Evidence suggests that PBSCs exhibit functions that are similar to bone marrow-derived hematopoietic stem cells[12] and that the number of mobilized PBSCs is equivalent to, or even higher than, the number of stem cells from normal bone marrow[13]. Stem cells have no well-defined morphological characteristics, but PBSC collection after granulocyte colony-stimulating factor mobilization can result in the acquisition of more abundant mononuclear and CD34+ cells. In addition, this method has advantages: it is easy to perform, there is no need to anesthetize the patient, there are less complications, and it is reproducible[14].
This study demonstrated that during a 28-month follow-up of 11 patients, TB gradually decreased in 9 (81.82%), ALB began to recover toward normal levels in 8 (72.73%), PT gradually increased in 10 (90.91%), ascitic fluid decreased in 7 (63.64%), appetite increased in 8 (72.73%), physical strength returned in 7 (63.64%), and abdominal distension was alleviated in 7 (63.64%). At 2 weeks, and at 1, 2 and 3 months postoperatively, the majority of clinical symptoms were obviously alleviated in all 37 patients with decompensated liver cirrhosis, indicating the safety of PBSC mobilization, collection, and transplantation. Three patients experienced decreased liver function and increased ALT and TB levels, which may have been caused by the use of a contrast agent in the visualization. In the present study, all patients had class C Child-Pugh liver disease, and so the use of contrast agent would likely worsen any liver function injury. However, this was a transient response, and liver function was regained after 1 week of symptomatic treatment. One patient died of massive hemorrhaging of the upper gastrointestinal tract caused by portal hypertension. The preliminary clinical findings of this study suggest that after autologous PBSC transplantation, clinical symptoms and liver function of patients with decompensated liver cirrhosis largely improved, indicating that autologous PBSC transplantation is feasible. However, not all patients gained improvement in liver function after surgery; it is possible that severe liver damage influences intrahepatic settling and differentiation of PBSCs.
It is extremely important that indices predictive of the transformation of liver cirrhosis into liver cancer be periodically monitored. To date, AFP is the most specific index of primary liver cancer[18], but increased serum AFP levels are also observed in some patients with liver cirrhosis or chronic liver diseases. At present, most hospitals consider an AFP level of > 400 ng/mL for 8 successive weeks the diagnostic criterion of hepatocellular carcinoma[19]. When patients maintain an AFP level of > 400 ng/mL for a long period, they should be diagnosed according to clinical symptoms. Therefore, it is difficult to distinguish between benign and malignant diseases simply according to AFP levels. In this situation, AFP variant detection is of clinical significance, particularly for AFP values of 20 to 400 ng/mL. A previous study demonstrated that when an AFP-L3% level of ≥10% was used as the diagnostic criterion of hepatocellular carcinoma, the sensitivity of AFP-L3% in the diagnosis of hepatocellular carcinoma was 91.1% and its specificity in differential diagnoses of chronic liver diseases was 92.9%. In addition, the mean AFP-L3 value and the percentage of AFP-L3–positive cases among patients with hepatocellular carcinoma were significantly higher than in patients with liver cirrhosis and patients with chronic hepatitis. These findings indicate that AFP-L3 detection can help differentiate patients with primary hepatocellular carcinoma from those with liver cirrhosis and chronic hepatitis. In particular, in the AFP <400 ng/mL group, when an AFP-L3% level of ≥10% is considered to be the diagnostic criterion of hepatocellular carcinoma, hepatocellular carcinoma can be distinguished from benign liver diseases. Therefore, in patients with low AFP-L3 levels, detection of AFP-L3 would be of important clinical value[20].
Tubercles are often observed on radiographs of patients with liver cirrhosis, and these patients are suspected to have liver cancer if there is also an increased AFP level. AFP-L3 can distinguish the tubercles in liver cirrhosis from the space-occupying lesions associated with liver cancer. Moreover, the use of AFP-L3 as the diagnostic criterion of liver cancer is not restrained by an AFP level of >400 ng/mL. An AFP-L3 level of ≥10% denotes a gradually increasing number of malignant liver cells, indicating a high probability of liver cell canceration. Evidence suggests that when the tubercle size in the hepatic lobe is <5 cm, stage 1 liver cancer is likely and carries 5- and 10-year survival rates of 64.8% and 46.3% after tumor removal, respectively. However, the 5- and 10-year survival rates are only 37.1% and 29.2%, respectively, when the tubercle size is >5 cm. In addition, a smaller tubercle size is associated with better efficacy after tumor removal. Therefore, early diagnosis of primary hepatocellular carcinoma will greatly increase the chances of a cure. Table 2 shows that there was no significant difference in AFP-L3% among patients with different AFP levels, indicating that AFP-L3% is not influenced by AFP level. For this reason, clinical detection of AFP-L3 may help to identify benign and malignant liver diseases, in particular to earlier diagnose liver cancer with a tubercle size of <5 cm and differentially diagnose small size liver cancer and the liver diseases presenting low AFP levels.
In the present study, all 44 patients were diagnosed with liver cirrhosis by color Doppler flow imaging and underwent PBSC transplantation. They were not diagnosed with hepatocellular carcinoma when discharged, but the AFP and AFP-L3% levels were periodically followed up, and an AFP-L3% level of >10% was considered to be a positive diagnostic criterion of hepatocellular carcinoma. During a 6-month follow-up period, 5 patients were lost because of a lack of correspondence address; complete data was obtained in 39 patients. No tubercles were observed by either type B ultrasonography or computed tomography of patients with AFP critical levels and AFP-L3–positive expression. Previous results demonstrated that AFP-L3 expression existed in the first 6 to 12 months prior to imaging examinations. The majority of patients with liver cirrhosis and increases in AFP-L3 expression are usually diagnosed with hepatocellular carcinoma within 3 to 18 months. Results from this study demonstrated that detection of AFP-L3 levels would help predict the occurrence of liver cancer when the characteristic space-occupying lesions of liver cancer have not been observed. It is necessary to follow up and closely monitor the disease progression of patients with AFP-L3% levels of ≥10%.
The transplantation delivery pathway was a major factor in the present study. The liver is an ideal transplant site for MSC treatment of liver diseases, and different transplantation delivery pathways influence the intrahepatic settling, growth, and differentiation of MSCs in different ways. MSCs transplanted via peripheral veins can reach the liver, but only after entrance into the systemic circulation, which reduces the amount of stem cells that can differentiate in the liver[14-15, 21]. To increase the amount of transplanted stem cells that can differentiate in the liver, the majority of studies in China used transplantation via the hepatic artery because this method is convenient and simple. However, the arterial blood flow is rapid and the arterial inner diameter is thin; neither property is conducive to the settling and embedment of transplanted stem cells. Some investigators performed intrahepatic transplantation of MSCs via the portal vein and concluded that isolated MSCs were still present in the ramuli of the portal vein and central vein of the hepatic lobule in liver specimens ex vivo 12 days postoperatively. These transplanted stem cells could be integrated into the recipient hepatic plate and differentiate into mature liver cells[16-17, 22]. In the present study, the serum AFP level transiently increased 2 months postoperatively in 11 patients, but AFP-L3 levels were not altered, indicating that AFP-L3 undergoes no obvious changes during the inflammatory pathological process. This is consistent with its specificity as a tumor marker.
There were two limitations to the present study. One was that the small sample size prevented accurate assessment of the relationship between PBSC transplantation and liver cancer. The other was that the observation period was short, preventing a full understanding of the relationships between PBSC transplantation, the intrahepatic microenvironment, and intrahepatic malignant biological phenotypes. Further studies will be needed to fully determine the efficacy and safety of PBSC transplantation via the portal vein.
In conclusion, the preliminary clinical results of the present study demonstrated that the clinical symptoms and liver function of patients with decompensated liver cirrhosis recovered to a certain degree after PBSC transplantation. Furthermore, the detection within a short time period of AFP-L3, a serological marker of liver cancer, did not indicate the presence of a malignant biological phenotype.

Acknowledgments: We are grateful to Xiaoxiang Li and Shu Han from the ZMKS International Cancer Therapy Biotechnologies Co., Ltd., for providing supports in cell isolation, detection of cell activity and flow cytometry of cellular immunological indices. We also thank Guokun Ao, Youming Tan from the Department of Interventional Radiology, the 309 Hospital of Chinese PLA for providing technique supports in cell transplantation.
Conflicts of interest: None declared.

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 (Edited by Zhang H, Lin XD/Su LL/Song LP)