Improved Liver Function in Patients with Liver Cirrhosis After ...

TRANSLATIONAL AND CLINICAL RESEARCH Improved Liver Function in Patients with Liver Cirrhosis After Autologous Bone Marrow Cell Infusion Therapy SHUJI TERAI,a TSUYOSHI ISHIKAWA,a KAORU OMORI,a KOJI AOYAMA,a YOSHIO MARUMOTO,a YOHEI URATA,a YUICHIROU YOKOYAMA,a KOICHI UCHIDA,a TAKAHIRO YAMASAKI,a YASUHIKO FUJII,b KIWAMU OKITA,a ISAO SAKAIDAa a

Department of Molecular Science & Applied Medicine (Gastroenterology & Hepatology), Yamaguchi University Graduate School of Medicine, Ube, Yamaguchi, Japan; bCenter of Regenerative Medicine and Cell Therapy, Yamaguchi University Hospital, Yamaguchi, Japan

Key Words. Liver cirrhosis • Regenerative medicine • Liver regeneration • Autologous bone marrow cell infusion Stem cell therapy • Clinical trial

ABSTRACT We here report nine liver cirrhosis (LC) patients that underwent autologous bone marrow cell infusion (ABMI) from the peripheral vein. Subjects were patients with LC with total bilirubin of less than 3.0 mg/dl, platelet count of more than 5 (1010/l), and no viable hepatocellular carcinoma on diagnostic imaging. Autologous bone marrow (BM; 400 ml) was isolated from the ilium under general anesthesia. Mononuclear cells (MNCs) were separated by cell washing and were infused via the peripheral vein. MNC characteristics were confirmed by fluorescence-activated cell sorting analysis (CD34, CD45, and c-kit). After ABMI therapy, liver function was monitored by blood examination for 24 weeks.

From 400 ml of BM, we obtained 7.81 ⴞ 0.98 ⴛ 109 MNCs. After washing, 5.20 ⴞ 0.63 ⴛ 109 MNCs were infused into patients with LC. Significant improvements in serum albumin levels and total protein were observed at 24 weeks after ABMI therapy (p < .05). Significantly improved Child-Pugh scores were seen at 4 and 24 weeks (p < .05). ␣-Fetoprotein and proliferating cell nuclear antigen (PCNA) expression in liver biopsy tissue was significantly elevated after ABMI therapy (p < .05). No major adverse effects were noted. In conclusion, ABMI therapy should be considered as a novel treatment for patients with decompensated LC. STEM CELLS 2006;24:2292–2298

INTRODUCTION

To confirm the potential of cell therapy using BMCs, we developed an in vivo mouse model (green fluorescent protein [GFP]/carbon tetrachloride [CCl4]) to monitor BMC differentiation into hepatocytes [5]. We demonstrated that the transplanted GFP-positive BMCs populated the damaged liver and differentiated into albumin-producing hepatocytes via hepatoblast intermediates in chronic liver injury induced by continuous administration of CCl4 [5]. Furthermore, BMC infusion elevated serum albumin levels, reduced liver fibrosis, and improved survival rate [6, 7]. Taken together, these results suggest that BMC infusion is a potentially effective treatment for patients in liver failure [8]. Recently, am Esch et al. reported that portal administration of autologous CD133⫹ BMCs accelerated liver regeneration [9]. However, there have been no reports on autologous BMC infusion (ABMI) in patients with LC. Here, we report nine patients with LC treated with ABMI therapy.

Liver cirrhosis (LC) is the end stage of chronic liver disease and is very difficult to treat. Currently, liver transplantation is one of the only effective therapies available to such patients. However, serious problems are associated with liver transplantation: lack of donors, surgical complications, rejection, and high cost. Regenerative therapies have the potential to provide minimally invasive procedures with few complications. The potential for stem cells in bone marrow (BM) to differentiate into hepatocytes and intestinal cells was recently confirmed through detection of Y chromosome-containing cells in samples from female recipients of BM cells (BMCs) from male donors [1–3]. BMC transplantation has been performed to treat hematological diseases, and several clinical studies have applied BMC injection to induce regeneration of myocardium and blood vessels [4]. Taken together, these findings suggest that BMCs are effective sources for regenerative liver therapy.

Correspondence: Shuji Terai, M.D., Ph.D., Department of Molecular Science & Applied Medicine (Gastroenterology & Hepatology), Yamaguchi University Graduate School of Medicine, Minami Kogushi 1-1-1, Ube, Yamaguchi 755-8505, Japan. Telephone: ⫹81-836-22-2241; Fax: ⫹81-836-22-2240; e-mail: [email protected]. Received November 4, 2005; accepted for publication June 10, 2006; first published online in STEM CELLS EXPRESS June 15, 2006. © AlphaMed Press 1066-5099/2006/$20.00/0 doi: 10.1634/stemcells.2005-0542

STEM CELLS 2006;24:2292–2298 www.StemCells.com

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Table 1. Patient characteristics Age (years) Gender Etiology MNC infusion date Ascites Coma Albumin (g/dl) Total bilirubin (mg/dl) Prothrombin time (%) Child-Pugh score Medication Therapy

Patient 1

Patient 2

Patient 3

Patient 4

Patient 5

Patient 6

Patient 7

Patient 8

Patient 9

69 Male HBV Nov. 14, 2003

59 Male HBV Feb. 20, 2004

65 Female Unknown Mar. 5, 2004

69 Male HCV May 7, 2004

63 Male HCV May 21, 2004

55 Male HBV Sept. 24, 2004

56 Male HCV Jan. 14, 2005

60 Male HCV July 29, 2005

61 Male HCV Aug. 19, 2005

Positive (2) None (1) 2.6 (3)

None (1) None (1) 2.4 (3)

None (1) None (1) 3.1 (2)



None (1) None (1) 3.0 (2)

None (1) None (1) 2.1 (3)



1.0 (1)

0.6 (1)

2.7 (2)

1.0 (1)

2.5 (2)

1.2 (1)

1.6 (1)

1.8 (1)

2.5 (2)

65 (2)

62 (2)

62 (2)

80 (1)

62.3 (2)

58 (2)

68 (2)

79.6 (2)

60.7 (2)

9

8

8

8

10

7

8

9

9

Aminoleban EN 1 pack/day Livact 3 pack/day Furosemide

Livact 3 pack/day

Aminoleban EN

Aminoleban EN

Aminoleban EN

Livact

Aminoleban EN

Aminoleban EN

Livact

3 pack/day

1 pack/day

2 pack/day

3 pack/day

1 pack/day

2 pack/day

3 pack/day

Furosemide

Furosemide

Furosemide

40 mg/day

80 mg/day

30 mg/day

Spironolactone 50 mg/day






Livact 3 pack/day Furosemide 20 mg/day Spironolactone 25 mg/day

40 mg/day

Number in parentheses indicates points in total Child-Pugh score. Abbreviations: HBV, hepatitis B virus; HCV, hepatitis C virus; MNC, mononuclear cell.

MATERIALS

AND

METHODS

Patients Eligible patients were between 18 and 75 years of age and had a clinical diagnosis of LC. The present subjects were patients with LC with total bilirubin of less than 3.0 mg/dl, a platelet count of more than 5 (1010/l), and no viable hepatocellular carcinoma on computed tomography (CT) and magnetic resonance imaging. Patients were excluded from the study if they had problems in organs other than the liver (e.g., the heart or lungs). Patients who were observed for the 24-week period are shown in Table 1. Written informed consent was obtained from all patients.

Follow-Up (Laboratory Data, Ultrasonography, and CT) The study protocol is shown in Figure 1. After ABMI therapy, patients were followed up every week for 4 weeks, and laboratory data were analyzed monthly for 24 weeks. During the study period, medication was unchanged before and after ABMI therapy. Patients who changed medication or those who consumed alcohol were treated as dropouts. During the study, patients did not receive antiviral therapies such as interferon, lamivudine, ribavirin, or granulocyte colony-stimulating factor (G-CSF). Primary outcomes were safety and feasibility of ABMI therapy. Recruitment started in October 2003, and the first patient began treatment on November 14, 2003. A Child-Pugh score (albumin, total bilirubin, prothromibin time activity, ascites, and encephalopathy) was used to evaluate the overall condition of patients with LC [10]. Livers were examined by abdominal ultrasonography and CT to evaluate ascites. Total protein was monitored as a marker of nutrition, and serum pro-collagen-III peptide (PIIIP) was monitored as a marker of liver fibrosis [11]. Levels of serum hepatocyte growth factor (HGF) were also analyzed [12–14]. www.StemCells.com

ABMI Therapy BM (400 ml) was harvested from the ilium according to standard procedures under general anesthesia and was collected in plastic bags containing heparin [15, 16]. After fat was removed from the tops of the bags, hydroethylstarch was added at a final concentration of 1%. After 40-minute incubation at room temperature, red blood cells were precipitated. We used an automated bench-top device (Cytomate; Takara Bio Inc, Otsu, Shiga, Japan, http://www.takara-bio.com; Nihon Kohden Corporation, Tokyo, http://www.nihonkohden.com), which is a functionally closed system, for washing and concentrating mononuclear cells (MNCs) [17]. Cell washing and concentration were ensured using a spinning membrane connected to a filter wash bag. The final concentrated and washed cell product was made up to a final volume of 105 ml. Five milliliters of the final cell product was subjected to trypan blue dye exclusion test, endotoxin test, and fluorescence-activated cell sorting analysis. CD34-, CD45-, and c-kit-positive cells were determined by flow cytometry (EPICS XL-MCL; Beckman Coulter, Inc., Fullerton, CA, http://www.beckmancoulter.com, 92834-310.). CD34, CD45, and c-kit antibodies were obtained from Dako (Shijou douri, Sakyoku, Kyoto, Japan, http://www.dako.com). At 6 – 8 hours after BM harvest, the final MNC preparation was administered via the peripheral vein. All protocols were approved by the Ethics Committee of Yamaguchi University, and written informed consent was obtained from all patients.

Immunohistochemical Analysis of Proliferating Cell Nuclear Antigen and ␣-Fetoprotein Expression in Liver Biopsy Tissue Before and After ABMI Therapy Liver biopsy samples before ABMI therapy and at 4 weeks after ABMI therapy were obtained by ultrasound-guided liver biopsy to evaluate liver condition. We obtained liver biopsy samples

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Figure 1. Study protocols. Abbreviations: ABMI, autologous bone marrow cell infusion; BM, bone marrow; CT, computed tomography; MNC, mononuclear cell.

Table 2. Characteristics of processed mononuclear cells (MNCs)

Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 Patient 6 Patient 7 Patient 8 Patient 9 Average SE

No. of isolated cells (ⴛ109)

No. of infused MNCs (ⴛ109)

CD45ⴙ (%)

CD45ⴙ, CD34ⴙ (%)

CD45ⴙ CD34ⴙ c-kitⴙ (%)

CD45ⴚ (%)

13.50 6.24 11.20 4.29 5.1 8.88 7.78 5.74 6.73 7.81 ⫾ 0.98

7.40 3.63 8.05 2.21 4.78 6.42 5.66 3.74 4.92 5.20 ⫾ 0.63

88.4 92.3 97.6 90.3 93.9 97.0 98.5 93.7 92.2 93.76 ⫾ 1.13

1.0 5.7 3.0 2.1 3.8 1.1 0.3 2.1 2.4 2.39 ⫾ 0.54

0.6 1.8 0.9 0.8 1.2 0.6 0.2 1.0 1.3 0.93 ⫾ 0.16

11.8 8.0 2.4 9.7 6.1 3.1 1.5 6.3 7.8 6.30 ⫾ 1.15

from three patients (patients 2, 8, and 9). We used an ALOKA ProSound SSD-5500 ultrasound transducer (ALOKA Co., Ltd., Tokyo, http://www2.aloka.co.jp) and a 16-guage biopsy needle (Super-Core II Biopsy Instrument; Medical Device Technologies, Inc., Gainesville, FL, http://www.interv.net). Immunohistochemical staining was performed on formalin-fixed, paraffinembedded liver specimens using the standard avidin-biotinperoxidase complex (ABC) method. Specimens were small and were thoroughly infiltrated by formalin. Briefly, 3-␮m tissue sections were deparaffinized in xylene and dehydrated in an alcohol series. Endogenous peroxidase was blocked with fresh 0.3% hydrogen peroxide in methanol for 30 minutes at room temperature, followed by microwave antigen retrieval for 6 minutes at 95°C in 10 mM sodium citrate buffer (pH 6.0). Normal goat serum (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) was applied for 20 minutes and removed. This method is the same as previously described [14, 18, 19]. ␣-Fetoprotein (AFP) and proliferating cell nuclear antigen (PCNA) antibodies were used to monitor hepatocyte proliferation [13, 18, 20]. Sections were incubated with antiAFP (1:500) (goat) and anti-PCNA (1:500) (goat) (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, http://www.scbt.com) overnight at 4°C in a humidified chamber. After washing three times in phosphate-buffered saline (PBS) (Nissui Pharmaceutical Co., Ltd., Tokyo, http://www.nissui-pharm.co.jp), sections

were incubated with biotin-conjugated secondary antibody in PBS for 150 minutes at room temperature and were reacted with ABC for 30 minutes. Positive reactions were developed for 1.5 minutes using PBS containing hydrogen peroxidase and 3,3⬘diaminobenzidine. We captured five areas (⫻100) and counted at least 1,000 random hepatocytes during evaluation of cells positive for AFP and PCNA staining. The labeling index (positive/negative cells) was calculated [14, 18, 19]. Differences in labeling index between each group were analyzed for significance by Student’s t test.

Statistical Analysis Changes in laboratory data from baseline (before ABMI therapy) to 4 or 24 weeks after ABMI therapy were analyzed. Values are shown as means ⫾ SE. Data were analyzed by analysis of variance with Fisher’s projected least significant difference test.

RESULTS Follow-Up Between October 2003 and March 2006, 12 patients with LC were recruited into the study, but two were subsequently excluded. The reasons for exclusion were as follows: one patient exhibited a tendency for severe bleeding, and the other patient

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Figure 2. Improved liver function after ABMI therapy. (A): Serum albumin, total protein, Child-Pugh score, PIIIP, and HGF during 24-week follow-up period after ABMI therapy. (B): Serum level of PIIIP and HGF after ABMI therapy. Average: average value. ⴙ indicates significant difference between baseline value and that at 24 weeks after ABMI therapy. # indicates significant difference between baseline value and that at 24 weeks after ABMI therapy. ⴱ indicates significant difference between each group (before, 4 weeks after, and 24 weeks after ABMI therapy). ⽧ indicates no significant differences between the data at 8 weeks, 4 weeks, and 1 day before ABMI therapy. (Normal ranges: albumin 3.7– 4.7 g/dl, total protein 6.8 – 8.3 g/dl, PIIIP 0.3– 0.8 U/ml, and HGF 0 – 0.39 ng/ml). Abbreviations: ABMI, autologous bone marrow cell infusion; HGF, hepatocyte growth factor; PIIIP, pro-collagen III peptide.

was diagnosed with gastric cancer. One patient with LC (etiology was alcohol) dropped out of the study because he continued to consume alcohol during the follow-up period. Thus, a total of nine patients were followed up to the end in the study. The characteristics of these patients are shown in Table 1. Patient medications are also shown in Table 1. Patients were administrated branched-chain amino acid granules, Livact Granule (Ajinomoto Co., Inc., Tokyo, http://www.ajinomoto.com), or Aminoleban EN (Otsuka Pharmaceutical Co., Ltd., Tokyo, http:// www.otsuka.co.jp), which contains a high branched-chain amino acid content, to improve the nutritional disorders associated with LC [21, 22]. Spironolactone and Furosemide were www.StemCells.com

used to control ascites. Medications were not changed during the study period, and no antiviral therapies were administered.

Isolation of BMCs and Cell Products from Patients We isolated 400 ml of autologous BM from the ilium. As shown in Table 2, we obtained 7.81 ⫾ 0.98 ⫻ 109 MNCs. After washing with Cytomate, 5.20 ⫾ 0.63 ⫻ 109 MNCs were administered. MNC viability was more than 90%. The characteristics of the cell product are shown in Table 2.

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Figure 3. Abdominal ultrasonography and CT for patient 1. (A): Abdominal ultrasonography before ABMI therapy. Arrows indicate severe ascites. (B): CT shows severe ascites before ABMI therapy. Arrows indicate ascites. (C): Abdominal ultrasonography showing decrease in ascites at 2 weeks after ABMI therapy. (D): CT showing improvement in ascites at 2 weeks after ABMI therapy. Abbreviations: ABMI, autologous bone marrow cell infusion; CT, computed tomography.

Changes in Laboratory Data After ABMI Therapy Data for serum albumin and total protein, as well as Child-Pugh scores, at 8 weeks, 4 weeks, and 1 day before ABMI therapy were not significantly different. As shown in Figure 2A, average serum albumin levels and total protein had significantly improved at 24 weeks after ABMI therapy (p ⬍ .05). Child-Pugh scores were used to evaluate overall liver function, and the average scores improved significantly at 4 and 24 weeks after ABMI therapy (p ⬍ .05). As shown in Figure 2B, serum levels of PIIIP also tended toward improvement. Levels of HGF were slightly elevated.

Improvement of Ascites After ABMI Therapy As shown in Figure 3, ascites improved after ABMI therapy. In patient 1, abdominal ultrasonography and CT showed that ascites had decreased at 2 weeks after ABMI therapy (Fig. 3C, 3D). The same trend was observed in patients 4, 5, 8, and 9.

AFP and PCNA Expression in Liver Biopsy Tissue After ABMI Therapy We analyzed AFP and PCNA expression in liver biopsy tissue before and after ABMI therapy (patients 2, 8, and 9). As shown in Figure 4A and 4B, we found that AFP and PCNA expression was significantly elevated at 4 weeks after ABMI therapy (p ⬍ .05).

Complications All nine patients exhibited fever (approximately 38°C) at 1 day after ABMI therapy, but fever was not seen after that. No adverse effects were noted in any of the present patients.

DISCUSSION We demonstrated the safety of isolation of autologous BM from patients with LC and the effects of ABMI therapy. When we

Figure 4. Immunohistochemical analysis of AFP and PCNA expression. (A): AFP expression before and at 4 weeks after ABMI therapy in patient 2. Arrows indicate AFP-positive cells. Labeling index includes data for three patients (patients 2, 8, and 9). An asterisk indicates significant difference between baseline value and that at 4 weeks after ABMI therapy (p ⬍ .05). (B): PCNA expression before and at 4 weeks after ABMI therapy in patient 2. Arrows indicate PCNA-positive cells. Labeling index includes data for three patients (patients 2, 8, and 9). An asterisk indicates significant difference between baseline value and that at 4 weeks after ABMI therapy (p ⬍ .05). Magnifications, ⫻100. Abbreviations: ABMI, autologous bone marrow cell infusion; AFP, ␣-fetoprotein; PCNA, proliferating cell nuclear antigen.

began this study, there were no data regarding whether sufficient numbers of BMCs could be obtained from patients with LC. Previous reports suggested that anesthesia is associated with an increased risk of morbidity and mortality [23, 24]. However, Ziser et al. reported that elective hernia surgery was safely performed under general anesthesia [25]. Thus, we selected isolation of BMC from the ilium under general anesthesia, and despite our concerns regarding adverse effects due to general anesthesia, the present nine patients exhibited no such complications. We safely isolated 400 ml of BM from the ilium, and as shown in Table 2, we obtained 7.81 ⫾ 0.91 ⫻ 109 MNCs (average). After preparation, we administered 5.20 ⫾ 0.63 ⫻ 109 MNCs via the peripheral vein (average). MNC viability was

Terai, Ishikawa, Omori et al. found to exceed 90%. The characteristics of infused MNCs are shown in Table 2. These results suggest that we obtained sufficient numbers of MNCs from the patients with LC enrolled in this study. In addition, we did not observe any major adverse effects due to isolation of BM and ABMI therapy. The changes of liver function in all nine patients after ABMI therapy are shown in Figure 2. As shown in Figure 2A, mean serum albumin levels, total protein levels, and Child-Pugh scores improved significantly after ABMI therapy (p ⬍ .05). Ascites also improved in patients 1, 4, 5, 8, and 9 (Fig. 3). Figure 4 shows a comparison of AFP and PCNA expression in liver biopsy samples before and after ABMI therapy. AFP and PCNA expression, which was used as a marker of hepatocyte proliferation, increased significantly after ABMI therapy (patients 2, 8, and 9). These results suggest that hepatocyte proliferation is induced by ABMI therapy. am Esch et al. reported that portal administration of autologous CD133⫹ BMCs accelerated liver regeneration and is a novel therapy to support hepatic resection [9]. In contrast to their study, the subjects of our study were patients with LC. We previously showed in an animal study that BMC infusion improved liver function and liver fibrosis [5–7]. The present data support our previous analysis. Because serum PIIIP levels are a marker of liver fibrosis [11], we monitored serum levels after ABMI therapy. As shown in Figure 2, average serum PIIIP levels were slightly decreased after ABMI therapy, thus suggesting improvement of liver fibrosis. Levels of HGF were also slightly elevated, but the change was not significant. We reported that fibroblast growth factor-2 is important in facilitating the differentiation of BMCs into hepatocytes but that HGF is not vital [26]. Data from human studies corresponded with our basic study. However, further study is needed to confirm the significance of growth factor. When we started the study, we believed that G-CSF injection would mobilize BMCs as a treatment for patients with LC.

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Our previous basic study using an animal model showed that mesenchymal cells would be more useful for liver regeneration [6, 7, 27]. Another group demonstrated the potential of mesenchymal stem cells in regenerative medicine [28]. We thus planned to perform mesenchymal cell infusion. On the other hand, spontaneous splenic rupture during peripheral blood stemcell mobilization in a healthy donor has been reported [29]. The subjects of our study were LC patients with splenomegaly by portal hypertension. We believe that the risk of splenic rupture by G-CSF is higher in patients with LC. Therefore, we obtained autologous BMCs from the ilium under general anesthesia. The characteristics of processed MNCs are shown in Table 2. This information is useful for characterizing useful BMCs for patients with LC. Gordon et al. recently showed that the CD34positive cell has the ability to improve liver function in liver disease [30], which may support our results. The present report evaluated ABMI therapy in patients with LC. Our results suggest that ABMI therapy is a candidate modality in the treatment of decompensated LC. To demonstrate the full therapeutic value of this protocol, more comprehensive long-term case-control clinical studies and/or randomized control studies are required.

ACKNOWLEDGMENTS We thank Dr. Yuzuru Sato, Hiroshi Nishina, and Naomi Kojima for their valuable assistance. This study was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (No. 163,90211, 165,90597) and for translational research from the Ministry of Health, Labor and Welfare (H14-trans-5, H17-Specai-015).

DISCLOSURES The authors indicate no potential conflicts of interest.

9

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