Share this article on:

Therapeutic Effects of Hepatocyte Transplantation on Hemophilia B

Tatsumi, Kohei1; Ohashi, Kazuo2,3,4,5; Shima, Midori1; Nakajima, Yoshiyuki2; Okano, Teruo3; Yoshioka, Akira1

doi: 10.1097/TP.0b013e31817b9160
Brief Reports

Hepatocyte transplantation offers an alternative therapeutic approach in the treatment of liver-related diseases. Hemophilia B is a bleeding disorder lacking factor IX (FIX) production in the liver, and achieving more than 1% coagulation activity results in significant improvement in the quality of life of the patients. The aim of this study was to investigate the efficacy of hepatocyte transplantation in the mouse model of hemophilia B. We transplanted isolated normal mouse hepatocytes into the liver of FIX knock-out mice. In some recipient mice, additional hepatocyte transplantations were performed 15 days after the first transplant. The recipient plasma FIX activities increased at 1% to 2% and persisted throughout the experimental period. An additional increase was achieved by the repeated transplantation. Close correlation between FIX messenger RNA levels of the liver and plasma FIX activity levels was observed. These results demonstrate that hepatocyte transplantation can provide therapeutic benefits in the treatment of hemophilia B.

1 Department of Pediatrics, Nara Medical University, Kashihara, Nara, Japan.

2 Department of Surgery, Nara Medical University, Kashihara, Nara, Japan.

3 Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, Shinjuku-ku, Tokyo, Japan.

4 Department of Gastroenterological Surgery Tokyo Women’s Medical University, Tokyo, Japan.

This work was supported by grants form AIDS Research from the Ministry of Health, Labor and Welfare of Japan (A.Y.) and grant-in-Aid (18591957) (K.O.) and Special Coordination Funds for Promoting Science and Technology (K.O., T.O.), and from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

5 Address correspondence to: Kazuo Ohashi, M.D., Ph.D., Institute of Advanced Biomedical Engineering and Science, Tokyo Women’s Medical University, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan.


Received 8 November 2007. Revision requested 11 December 2007.

Accepted 2 April 2008.

Hemophilia B, a recessive X-chromosome linked congenital bleeding disorder, is caused by a failure in the production of coagulation factor IX (FIX) (1). The only treatments that are currently available are the replacement therapy with FIX concentrates from plasma-derived or recombinant protein sources (2). This treatment modality is inefficient and expensive, because of the requirement of life-long and frequent intravenous infusion of FIX concentrates. Although the gene therapy has been actively studied over the past decade to establish a novel therapy that could provide longer acting and safer production of FIX (3), recent clinical trials have yet to conclusively shown long-term therapeutic benefits (4, 5). One potential approach that may provide the FIX producing ability in hemophilia B is a whole liver transplantation, because FIX is predominantly produced in the liver (6–8). However, the establishment of organ transplantation as a common therapy is hampered by a worldwide shortage of donor livers. Provided that some portion of the donated liver can be used for the isolation of individual hepatocytes, this donor shortage would no longer be a major issue. This is an important point, because the cell type responsible for synthesizing coagulation FIX is the hepatocyte (9). Therefore, a cell-based therapy using isolated hepatocytes could provide a therapeutic approach in the treatment of hemophilia B. Hepatocyte transplantation has been recently performed in several countries for various inherited disorders of hepatic metabolism and acute liver failure (10, 11). In bleeding disorder, hepatocyte transplantation was applied in the clinics by Dhawan et al. (12), who described therapeutic benefits in two patients suffering with congenital factor VII deficiency. Our group has recently shown applying a tissue engineering approach using primary hepatocytes could successfully provide therapeutic effects in hemophilia A mice (13). However, the effect of hepatocyte transplantation to treat hemophilia B has yet to be experimentally documented in animals or in the clinics to the best of our knowledge. For this reason, this study was designed to investigate the efficacy of hepatocyte transplantation on hemophilia B.

Hepatocytes were isolated from C57Bl/6 wild-type mice using a collagenase perfusion method as previously described (13–16). The recipient FIX knock-out (FIX-KO) mice, syngeneic to donor mice (17), were transplanted with the isolated hepatocytes (1.5×106 cells in 200 μL) into the liver through the inferior pole of the spleen (n=25). As an experimental control, several FIX-KO mice received sham operation (n=7). To avoid excessive surgical procedure-related bleeding, all FIX-KO mice received intraperitoneal injection of 0.5 mL pooled normal mouse plasma 30 min before abdominal surgery (18). All procedures were successfully carried out without any issues related to bleeding and all of the mice survived throughout the experimental period. At days 5, 10, and 15, some of the mice were killed for histologic and messenger RNA (mRNA) analyses (n=7, 5, and 4, at each time point, respectively). All sham-operated mice were killed at day 15.

Blood samples were periodically obtained from retro-orbital plexus of the experimental mice. After anticoagulated with 0.1 volume of 3.8% sodium citrate, blood samples were centrifuged, and plasma samples were stored at −80°C until being analyzed. The plasma FIX activity (FIX:C) was quantified by 1-stage clotting assay based on the activated partial thromboplastin time using human FIX-deficient plasma. Normal mouse plasma was used as FIX:C standard. Each measurement was reported after subtraction of the preoperational baseline FIX:C levels. As a result, FIX:C of recipient mice increased to more than 1% and were stably maintained throughout the experimental period (Fig. 1A). The FIX:C levels were significantly higher in the recipient mice when compared with the levels in the sham-operated mice at every time point examined. At day 5, recipient mice showed a small, but insignificant increase in plasma alanine aminotransferase after the transplantation (n=25) compared with the sham-operated mice (n=7). The slight increase in the alanine aminotransferase levels were found to be declined back toward baseline levels at day 10 (Fig. 1B). These results indicated that hepatocyte transplantation into hemophilia B mice could provide a therapeutic effect by producing FIX from the engrafted donor hepatocytes without significant liver injuries.



Histologic detection of transplanted and engrafted hepatocytes was performed by fluorescence in situ hybridization analysis using mouse Y-chromosome specific probe on sections of female FIX-KO recipient liver that received male hepatocytes. The presence of hepatocytes with Y-chromosome signals were confirmed, indicating the transplanted hepatocytes engrafted into the liver parenchyma (figure not shown). It is also important to note that any cell fusion events were not observed.

To enhance the therapeutic production of FIX in the recipient mice, a repeat transplantation of isolated hepatocytes was performed 15 days after the initial procedure in some recipients by infusing 1.2×106 hepatocytes into the upper pole of the spleen (n=4). The other remaining recipients (n=5) were examined with only a single transplantation procedure. As shown in Figure 2, the FIX:C values of the FIX-KO mice at day 25 (10 days after the second transplantation) were 0.94%±0.05% and 1.85%±0.09% in the single- and double-transplanted recipient mice, respectively (P=0.038). Similar increases in FIX:C were also observed at day 35 (20 days after the second transplantation) in the double-transplanted group. These data clearly demonstrated that increasing therapeutic effects could be obtained with a repeated transplantation.



We also examined whether the engrafted hepatocytes were capable of transcribing FIX mRNA in the recipient mouse livers. Because shunting of the hepatocytes into the lung had been described in the previous experimental studies (19), we also investigated FIX mRNA levels in the lung. Total RNA was extracted from liver, lung, and spleen. Total RNA (1 μg) was reverse transcribed, and the first-strand complementary DNA samples were subjected to quantitative real-time polymerase chain reaction amplification for mouse FIX gene and β-actin gene. Serial dilutions of complementary DNAs of normal mouse liver were used to generate the standard amplification curves. As shown in Figure 3(A), an abundant level of FIX mRNA was detected in the liver, with even higher mRNA expression detected in the livers manipulated with the repeated transplantation. No FIX mRNA signal was detected in the lungs in any of the mouse groups. Incremental expression of FIX was detected in the spleen of single and double hepatocyte transplanted mice, but the levels were markedly lower compared with the livers. We examined the relationship between the FIX:C levels and the liver FIX mRNA levels, and found a direct positive correlation between the two parameters (R 2=0.7214) (Fig. 3B).



Furthermore, we assessed the development of neutralizing antibodies against FIX (FIX inhibitor) by Bethesda method using plasma obtained at killing (20). Detectable levels (>0.5 Bethesda U/mL) of FIX inhibitor was not measured in any of recipient mice. This demonstrates that bioengineered FIX produced from the transplanted hepatocytes does not associate with the development of FIX inhibitors.

To investigate the long-term engraftment of hepatocytes, we performed an another set of single transplantation experiment for 12 weeks (n=6), and confirmed long-term persistency of the increased FIX activities at 0.92%±0.22%, 0.78%±0.22%, 0.78%±0.22%, and 0.83%±0.17% at week 2, 4, 8, and 12, respectively.

The present study confirmed the proof-concept feasibility of hepatocyte transplantation as an alternative therapy to treat hemophilia B. The functional engraftment of transplanted hepatocytes within the recipient livers was confirmed by fluorescence in situ hybridization analyses, FIX mRNA expression, and the secretion of functional FIX into the blood circulation. To acquire the proper hemostatic activity, synthesized coagulation FIX requires several posttranscriptional modification steps within the hepatocytes, including cleavage and removal the prepro leader sequence of 46 amino-acids, and γ-carboxylation of the first 12 glutamic acid residues (21). For this reason, primary hepatocytes would be more appropriate for transplantation to produce coagulation factors in hemophilia B than other possible types of genetically modified cells expressing FIX.

Previous studies have shown that engrafted hepatocytes within the livers are able to proliferate in response to the regeneration signals occurred by surgical hepatectomy or chronic liver injuries (22, 23). Using primary hepatocytes, our group has developed several innovative approaches to create a functional liver system under the kidney capsule or in subcutaneous locations (13, 15, 16, 24, 25), and we have clearly demonstrated that these ectopically engrafted hepatocytes also possess the ability for proliferation (13, 16, 26). This would be a significant benefit in the use of these hepatocytes, because most of the adult hemophilia B patients presented with chronic hepatitis B and/or C viral infection as a result of treatments with blood-borne contaminated plasma-derived FIX concentrates. Although portion of the transplanted hepatocytes would be infected with hepatitis viruses in the mean time, it would be reasonable to speculate that engrafted hepatocytes will proliferate and expand, which would further increase the therapeutic effects.

In conclusion, the present studies described the feasibility and safety of hepatocyte transplantation as a treatment modality for hemophilia B. Current therapies to treat hemophilia have been confounded with problems, and the present findings represent an important step toward establishing an alternative therapeutic approach for the treatment of not only hemophilia, but other similar genetic disorders affecting the liver.

Back to Top | Article Outline


The authors thank Yuichi Komai, Yuka Bessho, and Sanae Taminishi (Department of Pediatrics, Nara Medical University) for their technical assistance, and Dr. Frank Park (Medical College of Wisconsin) for his critical reading of the manuscript.

Back to Top | Article Outline


1. Bolton-Maggs PH, Pasi KJ. Haemophilias A and B. Lancet 2003; 361: 1801.
2. Manco-Johnson MJ, Abshire TC, Shapiro AD, et al. Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia. N Engl J Med 2007; 357: 535.
3. Nathwani AC, Davidoff AM, Tuddenham EG. Prospects for gene therapy of haemophilia. Haemophilia 2004; 10: 309.
4. Manno CS, Chew AJ, Hutchison S, et al. AAV-mediated factor IX gene transfer to skeletal muscle in patients with severe hemophilia B. Blood 2003; 101: 2963.
5. Manno CS, Pierce GF, Arruda VR, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med 2006; 12: 342.
6. Gordon FH, Mistry PK, Sabin CA, et al. Outcome of orthotopic liver transplantation in patients with haemophilia. Gut 1998; 42: 744.
7. Ko S, Tanaka I, Kanehiro H, et al. Preclinical experiment of auxiliary partial orthotopic liver transplantation as a curative treatment for hemophilia. Liver Transpl 2005; 11: 579.
8. Merion RM, Delius RE, Campbell DA, Jr, et al. Orthotopic liver transplantation totally corrects factor IX deficiency in hemophilia B. Surgery 1988; 104: 929.
9. Boost KA, Auth MK, Woitaschek D, et al. Long-term production of major coagulation factors and inhibitors by primary human hepatocytes in vitro: perspectives for clinical application. Liver Int 2007; 27: 832.
10. Fisher RA, Strom SC. Human hepatocyte transplantation: Worldwide results. Transplantation 2006; 82: 441.
11. Ohashi K, Park F, Kay MA. Hepatocyte transplantation: Clinical and experimental application. J Mol Med 2001; 79: 617.
12. Dhawan A, Mitry RR, Hughes RD, et al. Hepatocyte transplantation for inherited factor VII deficiency. Transplantation 2004; 78: 1812.
13. Ohashi K, Waugh JM, Dake MD, et al. Liver tissue engineering at extrahepatic sites in mice as a potential new therapy for genetic liver diseases. Hepatology 2005; 41: 132.
14. Berry MN, Friend DS. High-yield preparation of isolated rat liver parenchymal cells: A biochemical and fine structural study. J Cell Biol 1969; 43: 506.
15. Ohashi K, Kay MA, Kuge H, et al. Heterotopically transplanted hepatocyte survival depends on extracellular matrix components. Transplant Proc 2005; 37: 4587.
16. Ohashi K, Kay MA, Yokoyama T, et al. Stability and repeat regeneration potential of the engineered liver tissues under the kidney capsule in mice. Cell Transplant 2005; 14: 621.
17. Lin HF, Maeda N, Smithies O, et al. A coagulation factor IX-deficient mouse model for human hemophilia B. Blood 1997; 90: 3962.
18. Snyder RO, Miao C, Meuse L, et al. Correction of hemophilia B in canine and murine models using recombinant adeno-associated viral vectors. Nat Med 1999; 5: 64.
19. Schneider A, Attaran M, Gratz KF, et al. Intraportal infusion of 99mtechnetium-macro-aggregrated albumin particles and hepatocytes in rabbits: Assessment of shunting and portal hemodynamic changes. Transplantation 2003; 75: 296.
20. Kasper CK, Pool JG. Letter: Measurement of mild factor VIII inhibitors in Bethesda units. Thromb Diath Haemorrh 1975; 34: 875.
21. Arruda VR, Hagstrom JN, Deitch J, et al. Posttranslational modifications of recombinant myotube-synthesized human factor IX. Blood 2001; 97: 130.
22. Kokudo N, Ohashi K, Takahashi S, et al. Effect of 70% hepatectomy on DNA synthesis in rat hepatocyte isograft into the spleen. Transplant Proc 1994; 26: 3464.
23. Zhang H, Miescher-Clemens E, Drugas G, et al. Intrahepatic hepatocyte transplantation following subtotal hepatectomy in the recipient: A possible model in the treatment of hepatic enzyme deficiency. J Pediatr Surg 1992; 27: 312.
24. Ohashi K, Marion PL, Nakai H, et al. Sustained survival of human hepatocytes in mice: A model for in vivo infection with human hepatitis B and hepatitis delta viruses. Nat Med 2000; 6: 327.
25. Yokoyama T, Ohashi K, Kuge H, et al. In vivo engineering of metabolically active hepatic tissues in a neovascularized subcutaneous cavity. Am J Transplant 2006; 6: 50.
26. Ohashi K, Yokoyama T, Yamato M, et al. Engineering functional two- and three-dimensional liver systems in vivo using hepatic tissue sheets. Nat Med 2007; 13: 880.

Hepatocyte transplantation; Hemophilia B; Coagulation factor IX; Experimental transplantation

© 2008 Lippincott Williams & Wilkins, Inc.