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Selective Repopulation of Mice Liver after Fas-Resistant Hepatocyte Transplantation

Selective Repopulation of Mice Liver after Fas-Resistant Hepatocyte Transplantation Cell Transplantation, Vol. 10, pp. 353–361, 2001 0963-6897/01 $20.00 + .00 Printed in the USA. All rights reserved. Copyright  2001 Cognizant Comm. Corp. www.cognizantcommunication.com Selective Repopulation of Mice Liver After Fas-Resistant Hepatocyte Transplantation Masayuki Fujino,*‡ Xiao-Kang Li,* Yusuke Kitazawa,* Naoko Funeshima,* Lei Guo,* Torayuki Okuyama,† Takashi Amano,‡ Hiroshi Amemiya,* and Seiichi Suzuki* *Department of Experimental Surgery and Bioengineering and †Genetics, National Children’s Medical Research Center, Tokyo, Japan ‡Department of Zootechnical Science, Tokyo University of Agriculture, Tokyo, Japan Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopu- lation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 × 10 cells in female MRL-+/+ (wild- type mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intrave- nously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The patho- logic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver. Key words: Fas; Hepatocyte transplantation; Real-time quantitative PCR; sry gene INTRODUCTION planted hepatocytes do not reconstruct easily when the recipient liver is intact. In an attempt to increase the Hepatocyte transplantation has the advantage that it efficiency of hepatocyte transplantation, investigators can be performed with low risk on clinical patients, and induced hepatocellular regeneration by performing a hepatocytes obtained from a single donor can be trans- partial hepatectomy, which resulted in a proliferative planted into several recipients. Thus, there has been stimulus for transplanted hepatocytes as well as for increasing interest in hepatocyte transplantation as a po- the original hepatocytes (2,8,18). In a model recently tential therapeutic alternative for orthotopic liver trans- described for liver regeneration using transgenic mice plantation in certain clinical situations (6). that expressed a urokinase-type plasminogen activator, Ever since reproducible methods were developed for chronic stimulus by a hepatocyte injury allowed a repop- isolating viable hepatocytes, different sites for their im- ulation of syngenic donor cells of up to 80% in the plantation have been investigated for the efficacy of he- recipient liver after transplantation at relatively few he- patocyte transplantation in various animal models. Func- patocytes (15). Studies using Bcl-2 transgenic mice tionally active hepatocytes were detected in a liver after demonstrated that hepatocytes that were resistant to Fas- intraportal transplantation (10,14,17). Intrasplenic trans- mediated apoptosis repopulated in an anti-Fas antibody- plantation resulted in migration of hepatocytes into the treated normal liver, indicating a selective advantage of liver, where the donor cells were detected for a long the genetically engineered hepatocytes over resident he- period (4,5,14). Therefore, transplantation via the splenic patocytes for proliferation (11). or portal route is considered to be the most successful In the present study, we investigated whether hepato- method for hepatocyte transplantation. However, trans- Address correspondence to Xiao-Kang Li, M.D., Ph.D., Department of Experimental Surgery & Bioengineering, National Children’s Medical Research Center, 3-35-31 Taishido Setagaya-ku, Tokyo, 154-8509 Japan. Tel: 81-3-3414-8121; Fax: 81-3-3414-3208; E-mail: sri@nch.go.jp 353 354 FUJINO ET AL. cytes of lpr/lpr mice, which are completely resistant to der specific pathogen-free conditions in our animal facil- Fas-mediated apoptotic liver failure due to a lack of Fas ity. Animal care was in accordance with the guidelines expression (12), would repopulate in normal livers of of the National Children’s Medical Research Center. wild-type mice treated with sublethal doses of anti-Fas Reagent and Antibody antibody (Jo2). To assess transplantation efficiency, we performed a real-time quantitative PCR assay based on We purchased collagenase type I from Nitta Gelatin, TaqMan methodology to quantify the sry gene, the Y Ltd. (Osaka, Japan) and Williams’ E medium (WE) chromosome-specific genomic region, in total DNA so- from GIBCO BRL, Life Technologies, Inc. (Rockville, lutions prepared from a female recipient liver grafted MD). Hamster anti-mouse Fas monoclonal antibody with male hepatocytes. We discovered that Fas-resistant (Jo2) was purchased from Pharmingen (San Diego, CA). hepatocytes can repopulate selectively in a normal Hepatocyte Isolation and Transplantation mouse liver treated with Jo2. We isolated mouse hepatocytes using a modification MATERIALS AND METHODS of the method described by Berry et al. (1). In brief, we Mice anesthetized the mice with ether and exposed the portal MRL-+/+ (wild-type) and MRL-lpr/lpr (lpr) mice, vein with a ventral midline abdominal incision. We in- 8–10 weeks old, were purchased from CLER JAPAN, serted a fluid-filled 24-gauge catheter into the portal Inc. (Tokyo, Japan). All mice used were maintained un- vein and introduced 20 ml of 37°C perfusion buffer con- Figure 1. Illustration of the mouse sry (A) and β-actin (B) gene sequence and placement of PCR primers and TaqMan probe. REPOPULATION OF FAS-RESISTANT HEPATOCYTE TRANSPLANT 355 Figure 2. Amplification plot generated by the PE Biosystems prism model 7700 sequence detec- tion system. (A) The change in normalized reporter signal (∆ R ) versus cycle number, plotted in a linear fashion, illustrates the growth of the fluorescent signal over time as a result of probe cleav- age. Duplicate samples of male hepatocytes DNA are shown. The horizontal black line represents the target fluorescence threshold. (B) The same samples shown in (A) are graphed here as a logarithmic plot of ∆ R versus cycle number. The horizontal black line represents the target fluo- rescence threshold and is set at a level identical to that in (A). This graph clearly illustrates the exponential growth of amplicon (fluorescence) during the geometric phase of the PCR. taining 137 mM NaCl, 4.69 mM KCl, 1.17 mM g for 1 min. The hepatocytes were then resuspended in NaH PO , 0.65 mM MgSO 7H O, 1.0 mM EGTA, and pellets in WE. The viability of the cells, as quantified 2 4 4 2 10 mM HEPES, pH 7.2. This was followed by 25 ml of by trypan blue exclusion, varied between 80% and 90%. an enzyme solution (2 mg/ml collagenase type I, 66.7 Male hepatocytes resuspended in WE (5 × 10 /ml) at mM NaCl, 6.70 mM KCl, 4.7 6 mM CaCl 2H O, 100 a volume of 200 µ l were injected into the inferior tip of 2 2 mM HEPES, pH. 7.5), which then passed through the the spleens of female wild-type mice. We divided the liver. The liver was excised immediately, diced into mice into two groups: male lpr hepatocytes were trans- small pieces, and placed into 50 ml of WE. The digested planted into wild-type female mice treated with Jo2 anti- tissue was then filtered through a sterile 85-µ m nylon body (the experimental group), and the control group mesh. We centrifuged the cell suspension twice at 50 × without the antibody. The administration dose of Jo2 356 FUJINO ET AL. was 0.15 mg/kg at 1 day after hepatocyte transplanta- mediated dUTP-biotin nick end labeling (TUNEL) stain- tion, 0.3 mg/kg at 1 week after grafting, and 0.6 mg/kg ing method using an ApopTag Plus In Situ Apoptosis at 2 weeks. Detection Kit (Oncor, Gainthersburg, MD). We fixed cryosections in 10% neutral-buffered formalin in a Sampling coplin jar. We treated the sections in 2–3% hydrogen We sacrificed five mice from each group 1, 2, and 3 peroxide in PBS for 5 min at room temperature, and months after hepatocyte transplantation. We embedded then incubated them with a terminal deoxynucleotidyl the liver blocks up to 1 cm in OCT compound (Tissue- transferase (TdT) reaction mixture consisting of 38 µ l Tek, Elkhart, IN) and snap-froze them in isopentane; 6- reaction buffer and 16 µ l TdT enzyme in a humidified µ m frozen sections were then cut in a cryostat for DNA chamber at 37°C for 1 h. The reaction was terminated fragmentation analysis and immunohistology. A second with prewarmed stop/wash buffer for 30 min at 37°C. part of the liver was immediately snap-frozen for subse- We incubated the sections with a peroxidase-conjugated quent molecular analysis, and a third part was fixed in anti-digoxigenin antibody for 30 min at room tempera- 10% neutral buffered formalin for routine histology. We ture to visualize incorporated TdT. The sections were obtained the mouse sera 24 h after Jo2 injection for the washed three times in a coplin jar and incubated with transaminase measurement. DAB substrate solution for 3–6 min. We terminated the reaction by washing with H O, counterstaining with he- Liver DNA Extraction matoxylin, and mounting them. We lysed the liver tissue with 500 µ l of lysis buffer (Tris-buffered saline supplemented with 10% SDS and Histological Studies 20 mg/ml Tween 20). The lysates were incubated for 16 Liver tissues fixed in 10% phosphate-buffered forma- hat 55°C with 100 µ g/ml proteinase K, followed by lin were embedded in paraffin and their 5-µ m-thick sec- phenol/chloroform extraction. The tissue DNA was etha- tions were stained with hematoxylin-eosin (H&E) for nol precipitated, dried, and resuspended in Tris-EDTA standard microscopy. buffer (10 mM Tris, pH 7.5, 5 mM EDTA). Real-Time Quantitative PCR Analysis Determination of Serum Transaminase Activities We performed real-time quantitative PCR to detect We determined the serum activities of aspartate ami- the mice sry gene using gene-specific double-fluorescent notransferase (AST) using a Vision kit (Abbott, Park, labeled probes in a prism 7700 Sequence Detector (PE IL) according to the manufacturer’s protocol. Applied Biosystems, Foster City, CA). We used 6-car- boxy fluoreacein (FAM) as the 5′ fluorescent reporter, RESULTS and added tetramethylrhodamine (TAMRA) to the 3′ Real-Time Quantitative PCR Assay end as quencher. The following primers and probe se- A TaqMan probe-primer set was designed using quences were used to detect the sry gene: forward Primer Express Software (PE Biosystems 2x Universal primer: 5′-TGTACAACCTTCTGCAGTGGG-3′; re- verse primer: 5′-GCAAGACTGGAGTAGAGCTGCA-3′; and probe 5′-FAM-CAGGAACCCACATGCCATCAC ATACA-TAMRA-3′; β-actin, housekeeping gene, for- ward primer: 5′-CTAGGCACCAAGGTGTGAT-3′; re- verse primer: 5′-CAAACATGATCTGGGTCATC-3′; probe: 5′-FAM-TGGCACCACACCTTCTACAATGAG- TAMRA-3′. The amplification conditions were as fol- lows: 50°C for 2 min, 95°C for 15 min, then 63 cycles of 95°C for 30 s and 60°C for 1 min. Standards for the percentage of male DNA per female DNA were pre- pared by serial dilution to obtain the standard curve over 3log. The DNA isolated from the liver tissue of female mice transplanted with male hepatocytes was analyzed in duplicate in the real-time quantitative PCR. We com- pensated the data from amplification of the sry gene to Figure 3. Standard curve of the male mice liver DNA. A sam- the β-actin housekeeping gene. ple consisting of male mice DNA was serially diluted from 200 to 0.1 ng and run with the sry TaqMan probe and primer In Situ Assay for DNA Fragmentation set. Cycle number (C ) is plotted against the log concentration Our detection of apoptotic cells in the mice livers of the sample (dashed line). Linear regression analysis is was based on the terminal deoxynucleotidyl transferase- shown as a solid line. REPOPULATION OF FAS-RESISTANT HEPATOCYTE TRANSPLANT 357 Master Mix) to quantify the frequency of sry sequences centration of DNA remained constant. This was re- in transplanted liver tissue (Fig. 1). flected in the slope of the β-actin curve (R = 0.0002), The kinetics of the TaqMan reaction were illustrated even though the concentration of the sry sequence in- as amplification plots (Figs. 2A and B). The PE Biosys- creased regularly across the 3logs of the dynamic range tems Prism 7700 collected data from each of the 96 (R = 0.9966) tested. These data clearly revealed that the wells every 7 s throughout the entire PCR. The reporter amplicons did not interfere with one another. dye signal was normalized to the passive reference Histological Changes and Serum Transaminase (ROX) found in the TaqMan buffer, and a matrix algo- Activities of the Mice After Jo2 Administration rithm was used to compare it with a pure dye spectra Three administrations of Jo2 in the present protocol that was included with the instrument. The last three led to massive hepatocyte apoptosis in the wild-type points taken from the extension phase were averaged and plotted versus the cycle number. A threshold was mice, although no animals died by this treatment (data used to determine when the accumulation of fluorescent not shown). The serum AST concentrations were in- signals from the reporter dye was significantly greater creased 24 h after injections with Jo2 at doses of 0.15 than the background. This threshold value was in place mg/kg the first time and 0.3 mg/kg a second time. How- during the geometric phase of the PCR. Examples of ever, we observed no marked increase in the AST levels the sample amplification indicated a strong and robust when the second injection was administered at the same reaction with extreme precision between replicates. dose as the first. This indicated that the mice obtained A serial dilution (3log-fold) of a sample containing resistance to the antibody (Fig. 5A). A histological study 200 ng of male liver DNA was tested with the sry probe- of livers from the Jo2-treated mice showed severe hepa- primer set to authenticate the real-time PCR analysis of tocyte vacuolation, multifocal hepatocyte death, and an each run. The results were plotted on a log scale and increased number of inflammatory cells [Fig. 5B(e) and subjected to linear regression analysis (Fig. 3). The val- (f)], while there was slight damage after the second in- ues were linear over the 3log range tested (R = 0.9909), jection at the same Jo2 dose as the first time [Fig. with a negative slope of −1.445. These results indicated 5B(g)]. In addition, these findings were not seen in the an extremely efficient PCR that remained linear down lpr mice with the same treatment as mentioned above to 0.1 ng of male liver DNA. [Fig. 5B(h)]. The observations of the TUNEL staining Standard mixtures of male (from 0.01% to 20%) and samples were consistent with the H&E findings [Fig. female liver DNA were tested with the TaqMan probe- 5B(a)–(d)]. primer sets for both sry and β-actin sequences to extrap- Transplantation of Male lpr Hepatocytes to Wild-Type olate the sry gene frequency of the hepatocyte samples. Female Mice Both targets were plotted as C versus log (% male We transplanted 1 × 10 hepatocytes from male lpr DNA) (Fig. 4). Although each of these samples con- mice into wild-type female mice and treated them three tained different percentages of male DNA, the total con- times with Jo2. DNA isolated from the recipient mice livers was taken at 1, 2, and 3 months after transplanta- tion to quantify the posttransplantation cell reconstruc- tion. We detected the presence of sry in the liver geno- mic DNA by real-time quantitative PCR analysis. Table 1 and Figure 6 summarize the relative numbers of donor cells of the isolated DNA. The percentage of male hepa- tocyte DNA in the total DNA was 2.9 ± 0.9 (mean ± SE). DISCUSSION Hepatocyte transplantation is a potential alternative treatment for some cases of orthotopic liver transplanta- tion. Previous studies demonstrated that the proportion of donor hepatocytes engrafted into the livers of normal recipients was less than 0.5% (14). Others indicated that Figure 4. Compensation of sry and β-actin results in identical a partial hepatectomy led to proliferation of the trans- samples of standard liver DNA mixtures. Individual samples planted hepatocytes, resulting in a slightly higher en- ranging from 20 to 0.01% male liver DNA are plotted as cycle graftment of donor cells (8,18). number (C ) against log of the concentration. Linear regression analysis is shown for each gene target. Fas, a type II TNF family receptor, induces intracel- 358 FUJINO ET AL. Figure 5. Histological findings and serum transaminase activities in the Jo2-treated mice. (A) AST concentrations were increased after 24 h when injected Jo2 at a dose of 0.15 mg/kg at the first time and 0.3 mg/kg at the second time. However, there was no dramatic change in AST when injecting the same dose as the first time, indicating the mice appeared to acquire resistance against the antibody. (B) Histological study of the liver from Jo2-treated mice showed a severe hepatocyte vacuolation, multifocal hepatocyte death, and increased number of inflammatory cells (e, f), while there was slight damage after the second injection of the Jo2 antibody at the same dose as the first time in normal mice (g), but not in lpr mice (h). The observation in TUNEL staining sample is consistent with H&E findings in a different manner (a–d). REPOPULATION OF FAS-RESISTANT HEPATOCYTE TRANSPLANT 359 Table 1. Hepatocyte Transplantation in Jo2-Treated Animals Ratio of Transplanted Cell DNA Treatment % Mean ± SE Mean ± SE Jo2 (anti-Fas antibody) 1 month E-1-1 3.3 E-1-2 1.3 E-1-3 1.6 1.5 ± 0.5 E-1-4 0.4 ] E-1-5 1.1 2 months E-2-1 2.8 E-2-2 6.9 E-2-3 3.6 3.1 ± 1.1 E-2-4 1.0 ] E-2-5 1.1 3 months E-3-1 1.2 2.9 ± 0.9 E-3-2 1.4 ] E-3-3 4.2 2.7 ± 0.7 E-3-4 4.4 ] E-3-5 2.2 Saline (control) 1 month C-1-1 0.2 C-1-2 0.1 C-1-3 0.1 ] 0.1 ± 0.03 C-1-4 0.03 C-1-5 0.03 2 months C-2-1 0.1 C-2-2 0.1 C-2-3 0.1 0.1 ± 0.01 C-2-4 0.1 ] C-2-5 0.1 3 months C-3-1 0.1 0.1 ± 0.03 C-3-2 0.1 ] C-3-3 0.1 0.1 ± 0.3 C-3-4 0.1 ] C-3-5 0.3 lular apoptotic signals through the caspase cascade. In- liver that contains approximately 1 × 10 hepatocytes. jection of an agonistic anti-Fas antibody (Jo2) via the The repopulation in the experimental group resulted tail vein into mice resulted in hepatitis accompanied by from the specific resistance of lpr hepatocytes to Fas- Fas-mediated apoptosis. In our experimental setting, we mediated apoptosis. In the control group, as expected, induced Fas-mediated hepatocyte apoptosis in vivo to the livers of mice transplanted with lpr hepatocytes but stimulate hepatocellular regeneration. A previous study without Jo2 treatments were not repopulated by these indicated that a sublethal dose of Jo2 kills approximately cells (Table 1). This result is consistent with the absence one third of the resident hepatocytes. We assumed that of repopulation of FAH−/− mice livers transplanted with both apoptosis-sensitive resident hepatocytes and apop- FAH+/+ hepatocytes and treated with NTBC (13). In tosis-resistant lpr hepatocytes participate in the regenera- this study, the administration of Jo2 after transplantation 6 6 tive process. We injected 1 × 10 lpr hepatocytes into a of 1 × 10 lpr hepatocytes resulted in repopulation of the recipient spleen, where we expected approximately 20% donor hepatocytes at 6.9% in the recipient livers. This (2 × 10 cells) of the cells to redistribute and survive in result was 70-fold higher than that obtained with infu- the liver (14,15). As expected, the number of repopulat- sion of the same number of hepatocytes with control ing lpr hepatocytes after three treatments of Jo2 was saline. 5 3 5 2 × 10 × (3/2) = 6.75 × 10 . These results correspond to Our results yielded evidence that lpr-derived hepato- the optimal percentage of 0.68% for a normal mouse cytes would contribute to repopulation in Fas-positive 360 FUJINO ET AL. assessed on an automate sequenator. A highly sensitive and reproducible method with a rapid turnaround time is required, especially in a clinical setting. We demonstrated in this study that there was no de- tectable signal when genomic male DNA was tested in the control female hepatocytes, thus indicating the speci- ficity of this primer pair and the fluorescent TaqMan probe set for the sry gene. In addition to the favorable comparison with the technique of quantitative competi- tive PCR, this new real-time PCR approach has several advantages over the myriad of other techniques that have been used to assess gene transfer or gene correc- tion. Furthermore, the potential extension of this meth- Figure 6. Effects of Jo2-induced regeneration of donor hepa- odology to RT-PCR cannot be overestimated. Such an tocytes transplantation. Mice were given Jo2 or normal saline accurate and sensitive method has the potential to mea- 1 day after hepatocyte transplantation. Mice were sacrificed sure any biologic system. 1–3 months after transplantation. The data show the percent- age of mouse sry gene in five recipients. The data are pre- In conclusion, we demonstrated that transplanted lpr sented as mean ± SE. hepatocytes repopulated significantly in a normal liver after selective destruction of the host hepatocytes by three administrations of the agonistic anti-Fas antibody hepatocytes of a normal liver when treated three times at sublethal doses. Estimation of the sry gene by quanti- with anti-Fas antibody at sublethal doses. Similar ap- tative real-time PCR is useful for defining the repopula- proaches using transgenic mice with bcl-2 genes or bcl- tive efficiency of transplanted male hepatocytes in a fe- 2-transduced hepatocytes using an adeno-associated vi- male liver. rus or lentivirus could allow reconstitution of a large ACKNOWLEDGMENTS: We thank Drs. H. Kimura and S. number of these hepatocytes in an anti-Fas antibody- Enosawa for their comments and useful suggestions. This study was supported by a Grant for Organized Research Com- treated recipient (7,11,16). An ideal repopulation proto- bination System from the Science and Technology Agency in col would also include the use of well-known hepato- Japan. toxic drugs or apoptosis-inducing agents to eliminate recipient hepatocytes and the use of anti-apoptotic REFERENCES gene-encoding donor cells (3). Our results provide im- 1. Berry, M. N.; Friend, D. S. High-yield preparation of iso- portant evidence that replacement of intact hepatocytes lated rat liver parenchymal cells: A biochemical and fine with genetically modified cells is possible. structural study. J. Cell. Biol. 43:506–520; 1969. A new technique for quantifying PCR products in a 2. Borel Rinkes, I. H.; Bijma, A.; Kazemier, G.; Sinaasappel, M.; Valerio, D.; Terpstra, O. T. Proliferative response of real-time operation was recently developed. This tech- hepatocytes transplanted into spleen or solid support. J. nique, developed by TaqMan Chemistry, enables visual- Surg. Res. 56:417–423; 1994. ization of the accumulation of the target and the kinetics 3. Dbaibo, G. S.; Hannun, Y. A. Cytokine response modifier of each reaction (9). The signal generated is not only A (CrmA): A strategically deployed viral weapon. Clin. highly specific, but also directly proportional to the Immunol. Immunopathol. 86:134–140; 1998. 4. Gupta, S.; Aragona, E.; Vemuru, R. P.; Bhargava, K. K.; number of amplicons produced at each cycle, thus pro- Burk, R. D.; Chowdhury, J. R. Permanent engraftment and viding a means of quantifying the PCR products. In this function of hepatocytes delivered to the liver: Implications study, we employed a relatively new instrument, the PE for gene therapy and liver repopulation. Hepatology 14: Biosystems Prism Model 7700 sequence detection sys- 144–149; 1991. tem, to detect the signal generated by the TaqMan assay. 5. Gupta, S.; Vemuru, R. P.; Lee, C. D.; Yerneni, P. R.; Ara- gona, E.; Burk, R. D. 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Surg. 27:312–316; 1992. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Cell Transplantation SAGE

Selective Repopulation of Mice Liver after Fas-Resistant Hepatocyte Transplantation

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Abstract

Cell Transplantation, Vol. 10, pp. 353–361, 2001 0963-6897/01 $20.00 + .00 Printed in the USA. All rights reserved. Copyright  2001 Cognizant Comm. Corp. www.cognizantcommunication.com Selective Repopulation of Mice Liver After Fas-Resistant Hepatocyte Transplantation Masayuki Fujino,*‡ Xiao-Kang Li,* Yusuke Kitazawa,* Naoko Funeshima,* Lei Guo,* Torayuki Okuyama,† Takashi Amano,‡ Hiroshi Amemiya,* and Seiichi Suzuki* *Department of Experimental Surgery and Bioengineering and †Genetics, National Children’s Medical Research Center, Tokyo, Japan ‡Department of Zootechnical Science, Tokyo University of Agriculture, Tokyo, Japan Hepatocyte transplantation has been proposed as a potential therapeutic method to treat irreversible liver failure and inherited hepatic disorders, although transplanted cells do not easily reconstruct the liver tissue under intact conditions. This study was aimed at modulating the recipient liver conditions to promote repopu- lation of the liver after hepatocyte transplantation. Hepatocytes isolated from male MRL-lpr/lpr (lpr) mice with a mutation of Fas antigen were transplanted in a number of 1 × 10 cells in female MRL-+/+ (wild- type mice) by intrasplenic injection. An agonistic anti-Fas antibody (0.15 mg/kg) was administered intrave- nously 24 h after cell transplantation. We also administrated the antibody at 0.3 mg/kg 1 week after grafting and at 0.6 mg/kg 2 weeks after transplantation. The liver specimens were taken at different time intervals for histological examination. The reconstructed male lpr hepatocytes in the female wild-type mice were determined by a real-time quantitative PCR assay using the primers and probe for the sry gene. The patho- logic findings of the recipient livers after treatment with anti-Fas antibody revealed a large number of apoptotic hepatocytes. The grafted lpr hepatocytes were observed to reconstruct as much as 6.9% of the recipient liver in the anti-Fas antibody-treated group 3 months after transplantation. In contrast, we observed the transplanted cells at lower than 0.1% in the nontreated livers. These findings demonstrated that repeated induction of apoptosis in recipient hepatocytes shifts the environment of the liver to a regenerative condition. This method may be useful to promote the reconstruction of transplanted hepatocytes in a recipient liver. Key words: Fas; Hepatocyte transplantation; Real-time quantitative PCR; sry gene INTRODUCTION planted hepatocytes do not reconstruct easily when the recipient liver is intact. In an attempt to increase the Hepatocyte transplantation has the advantage that it efficiency of hepatocyte transplantation, investigators can be performed with low risk on clinical patients, and induced hepatocellular regeneration by performing a hepatocytes obtained from a single donor can be trans- partial hepatectomy, which resulted in a proliferative planted into several recipients. Thus, there has been stimulus for transplanted hepatocytes as well as for increasing interest in hepatocyte transplantation as a po- the original hepatocytes (2,8,18). In a model recently tential therapeutic alternative for orthotopic liver trans- described for liver regeneration using transgenic mice plantation in certain clinical situations (6). that expressed a urokinase-type plasminogen activator, Ever since reproducible methods were developed for chronic stimulus by a hepatocyte injury allowed a repop- isolating viable hepatocytes, different sites for their im- ulation of syngenic donor cells of up to 80% in the plantation have been investigated for the efficacy of he- recipient liver after transplantation at relatively few he- patocyte transplantation in various animal models. Func- patocytes (15). Studies using Bcl-2 transgenic mice tionally active hepatocytes were detected in a liver after demonstrated that hepatocytes that were resistant to Fas- intraportal transplantation (10,14,17). Intrasplenic trans- mediated apoptosis repopulated in an anti-Fas antibody- plantation resulted in migration of hepatocytes into the treated normal liver, indicating a selective advantage of liver, where the donor cells were detected for a long the genetically engineered hepatocytes over resident he- period (4,5,14). Therefore, transplantation via the splenic patocytes for proliferation (11). or portal route is considered to be the most successful In the present study, we investigated whether hepato- method for hepatocyte transplantation. However, trans- Address correspondence to Xiao-Kang Li, M.D., Ph.D., Department of Experimental Surgery & Bioengineering, National Children’s Medical Research Center, 3-35-31 Taishido Setagaya-ku, Tokyo, 154-8509 Japan. Tel: 81-3-3414-8121; Fax: 81-3-3414-3208; E-mail: sri@nch.go.jp 353 354 FUJINO ET AL. cytes of lpr/lpr mice, which are completely resistant to der specific pathogen-free conditions in our animal facil- Fas-mediated apoptotic liver failure due to a lack of Fas ity. Animal care was in accordance with the guidelines expression (12), would repopulate in normal livers of of the National Children’s Medical Research Center. wild-type mice treated with sublethal doses of anti-Fas Reagent and Antibody antibody (Jo2). To assess transplantation efficiency, we performed a real-time quantitative PCR assay based on We purchased collagenase type I from Nitta Gelatin, TaqMan methodology to quantify the sry gene, the Y Ltd. (Osaka, Japan) and Williams’ E medium (WE) chromosome-specific genomic region, in total DNA so- from GIBCO BRL, Life Technologies, Inc. (Rockville, lutions prepared from a female recipient liver grafted MD). Hamster anti-mouse Fas monoclonal antibody with male hepatocytes. We discovered that Fas-resistant (Jo2) was purchased from Pharmingen (San Diego, CA). hepatocytes can repopulate selectively in a normal Hepatocyte Isolation and Transplantation mouse liver treated with Jo2. We isolated mouse hepatocytes using a modification MATERIALS AND METHODS of the method described by Berry et al. (1). In brief, we Mice anesthetized the mice with ether and exposed the portal MRL-+/+ (wild-type) and MRL-lpr/lpr (lpr) mice, vein with a ventral midline abdominal incision. We in- 8–10 weeks old, were purchased from CLER JAPAN, serted a fluid-filled 24-gauge catheter into the portal Inc. (Tokyo, Japan). All mice used were maintained un- vein and introduced 20 ml of 37°C perfusion buffer con- Figure 1. Illustration of the mouse sry (A) and β-actin (B) gene sequence and placement of PCR primers and TaqMan probe. REPOPULATION OF FAS-RESISTANT HEPATOCYTE TRANSPLANT 355 Figure 2. Amplification plot generated by the PE Biosystems prism model 7700 sequence detec- tion system. (A) The change in normalized reporter signal (∆ R ) versus cycle number, plotted in a linear fashion, illustrates the growth of the fluorescent signal over time as a result of probe cleav- age. Duplicate samples of male hepatocytes DNA are shown. The horizontal black line represents the target fluorescence threshold. (B) The same samples shown in (A) are graphed here as a logarithmic plot of ∆ R versus cycle number. The horizontal black line represents the target fluo- rescence threshold and is set at a level identical to that in (A). This graph clearly illustrates the exponential growth of amplicon (fluorescence) during the geometric phase of the PCR. taining 137 mM NaCl, 4.69 mM KCl, 1.17 mM g for 1 min. The hepatocytes were then resuspended in NaH PO , 0.65 mM MgSO 7H O, 1.0 mM EGTA, and pellets in WE. The viability of the cells, as quantified 2 4 4 2 10 mM HEPES, pH 7.2. This was followed by 25 ml of by trypan blue exclusion, varied between 80% and 90%. an enzyme solution (2 mg/ml collagenase type I, 66.7 Male hepatocytes resuspended in WE (5 × 10 /ml) at mM NaCl, 6.70 mM KCl, 4.7 6 mM CaCl 2H O, 100 a volume of 200 µ l were injected into the inferior tip of 2 2 mM HEPES, pH. 7.5), which then passed through the the spleens of female wild-type mice. We divided the liver. The liver was excised immediately, diced into mice into two groups: male lpr hepatocytes were trans- small pieces, and placed into 50 ml of WE. The digested planted into wild-type female mice treated with Jo2 anti- tissue was then filtered through a sterile 85-µ m nylon body (the experimental group), and the control group mesh. We centrifuged the cell suspension twice at 50 × without the antibody. The administration dose of Jo2 356 FUJINO ET AL. was 0.15 mg/kg at 1 day after hepatocyte transplanta- mediated dUTP-biotin nick end labeling (TUNEL) stain- tion, 0.3 mg/kg at 1 week after grafting, and 0.6 mg/kg ing method using an ApopTag Plus In Situ Apoptosis at 2 weeks. Detection Kit (Oncor, Gainthersburg, MD). We fixed cryosections in 10% neutral-buffered formalin in a Sampling coplin jar. We treated the sections in 2–3% hydrogen We sacrificed five mice from each group 1, 2, and 3 peroxide in PBS for 5 min at room temperature, and months after hepatocyte transplantation. We embedded then incubated them with a terminal deoxynucleotidyl the liver blocks up to 1 cm in OCT compound (Tissue- transferase (TdT) reaction mixture consisting of 38 µ l Tek, Elkhart, IN) and snap-froze them in isopentane; 6- reaction buffer and 16 µ l TdT enzyme in a humidified µ m frozen sections were then cut in a cryostat for DNA chamber at 37°C for 1 h. The reaction was terminated fragmentation analysis and immunohistology. A second with prewarmed stop/wash buffer for 30 min at 37°C. part of the liver was immediately snap-frozen for subse- We incubated the sections with a peroxidase-conjugated quent molecular analysis, and a third part was fixed in anti-digoxigenin antibody for 30 min at room tempera- 10% neutral buffered formalin for routine histology. We ture to visualize incorporated TdT. The sections were obtained the mouse sera 24 h after Jo2 injection for the washed three times in a coplin jar and incubated with transaminase measurement. DAB substrate solution for 3–6 min. We terminated the reaction by washing with H O, counterstaining with he- Liver DNA Extraction matoxylin, and mounting them. We lysed the liver tissue with 500 µ l of lysis buffer (Tris-buffered saline supplemented with 10% SDS and Histological Studies 20 mg/ml Tween 20). The lysates were incubated for 16 Liver tissues fixed in 10% phosphate-buffered forma- hat 55°C with 100 µ g/ml proteinase K, followed by lin were embedded in paraffin and their 5-µ m-thick sec- phenol/chloroform extraction. The tissue DNA was etha- tions were stained with hematoxylin-eosin (H&E) for nol precipitated, dried, and resuspended in Tris-EDTA standard microscopy. buffer (10 mM Tris, pH 7.5, 5 mM EDTA). Real-Time Quantitative PCR Analysis Determination of Serum Transaminase Activities We performed real-time quantitative PCR to detect We determined the serum activities of aspartate ami- the mice sry gene using gene-specific double-fluorescent notransferase (AST) using a Vision kit (Abbott, Park, labeled probes in a prism 7700 Sequence Detector (PE IL) according to the manufacturer’s protocol. Applied Biosystems, Foster City, CA). We used 6-car- boxy fluoreacein (FAM) as the 5′ fluorescent reporter, RESULTS and added tetramethylrhodamine (TAMRA) to the 3′ Real-Time Quantitative PCR Assay end as quencher. The following primers and probe se- A TaqMan probe-primer set was designed using quences were used to detect the sry gene: forward Primer Express Software (PE Biosystems 2x Universal primer: 5′-TGTACAACCTTCTGCAGTGGG-3′; re- verse primer: 5′-GCAAGACTGGAGTAGAGCTGCA-3′; and probe 5′-FAM-CAGGAACCCACATGCCATCAC ATACA-TAMRA-3′; β-actin, housekeeping gene, for- ward primer: 5′-CTAGGCACCAAGGTGTGAT-3′; re- verse primer: 5′-CAAACATGATCTGGGTCATC-3′; probe: 5′-FAM-TGGCACCACACCTTCTACAATGAG- TAMRA-3′. The amplification conditions were as fol- lows: 50°C for 2 min, 95°C for 15 min, then 63 cycles of 95°C for 30 s and 60°C for 1 min. Standards for the percentage of male DNA per female DNA were pre- pared by serial dilution to obtain the standard curve over 3log. The DNA isolated from the liver tissue of female mice transplanted with male hepatocytes was analyzed in duplicate in the real-time quantitative PCR. We com- pensated the data from amplification of the sry gene to Figure 3. Standard curve of the male mice liver DNA. A sam- the β-actin housekeeping gene. ple consisting of male mice DNA was serially diluted from 200 to 0.1 ng and run with the sry TaqMan probe and primer In Situ Assay for DNA Fragmentation set. Cycle number (C ) is plotted against the log concentration Our detection of apoptotic cells in the mice livers of the sample (dashed line). Linear regression analysis is was based on the terminal deoxynucleotidyl transferase- shown as a solid line. REPOPULATION OF FAS-RESISTANT HEPATOCYTE TRANSPLANT 357 Master Mix) to quantify the frequency of sry sequences centration of DNA remained constant. This was re- in transplanted liver tissue (Fig. 1). flected in the slope of the β-actin curve (R = 0.0002), The kinetics of the TaqMan reaction were illustrated even though the concentration of the sry sequence in- as amplification plots (Figs. 2A and B). The PE Biosys- creased regularly across the 3logs of the dynamic range tems Prism 7700 collected data from each of the 96 (R = 0.9966) tested. These data clearly revealed that the wells every 7 s throughout the entire PCR. The reporter amplicons did not interfere with one another. dye signal was normalized to the passive reference Histological Changes and Serum Transaminase (ROX) found in the TaqMan buffer, and a matrix algo- Activities of the Mice After Jo2 Administration rithm was used to compare it with a pure dye spectra Three administrations of Jo2 in the present protocol that was included with the instrument. The last three led to massive hepatocyte apoptosis in the wild-type points taken from the extension phase were averaged and plotted versus the cycle number. A threshold was mice, although no animals died by this treatment (data used to determine when the accumulation of fluorescent not shown). The serum AST concentrations were in- signals from the reporter dye was significantly greater creased 24 h after injections with Jo2 at doses of 0.15 than the background. This threshold value was in place mg/kg the first time and 0.3 mg/kg a second time. How- during the geometric phase of the PCR. Examples of ever, we observed no marked increase in the AST levels the sample amplification indicated a strong and robust when the second injection was administered at the same reaction with extreme precision between replicates. dose as the first. This indicated that the mice obtained A serial dilution (3log-fold) of a sample containing resistance to the antibody (Fig. 5A). A histological study 200 ng of male liver DNA was tested with the sry probe- of livers from the Jo2-treated mice showed severe hepa- primer set to authenticate the real-time PCR analysis of tocyte vacuolation, multifocal hepatocyte death, and an each run. The results were plotted on a log scale and increased number of inflammatory cells [Fig. 5B(e) and subjected to linear regression analysis (Fig. 3). The val- (f)], while there was slight damage after the second in- ues were linear over the 3log range tested (R = 0.9909), jection at the same Jo2 dose as the first time [Fig. with a negative slope of −1.445. These results indicated 5B(g)]. In addition, these findings were not seen in the an extremely efficient PCR that remained linear down lpr mice with the same treatment as mentioned above to 0.1 ng of male liver DNA. [Fig. 5B(h)]. The observations of the TUNEL staining Standard mixtures of male (from 0.01% to 20%) and samples were consistent with the H&E findings [Fig. female liver DNA were tested with the TaqMan probe- 5B(a)–(d)]. primer sets for both sry and β-actin sequences to extrap- Transplantation of Male lpr Hepatocytes to Wild-Type olate the sry gene frequency of the hepatocyte samples. Female Mice Both targets were plotted as C versus log (% male We transplanted 1 × 10 hepatocytes from male lpr DNA) (Fig. 4). Although each of these samples con- mice into wild-type female mice and treated them three tained different percentages of male DNA, the total con- times with Jo2. DNA isolated from the recipient mice livers was taken at 1, 2, and 3 months after transplanta- tion to quantify the posttransplantation cell reconstruc- tion. We detected the presence of sry in the liver geno- mic DNA by real-time quantitative PCR analysis. Table 1 and Figure 6 summarize the relative numbers of donor cells of the isolated DNA. The percentage of male hepa- tocyte DNA in the total DNA was 2.9 ± 0.9 (mean ± SE). DISCUSSION Hepatocyte transplantation is a potential alternative treatment for some cases of orthotopic liver transplanta- tion. Previous studies demonstrated that the proportion of donor hepatocytes engrafted into the livers of normal recipients was less than 0.5% (14). Others indicated that Figure 4. Compensation of sry and β-actin results in identical a partial hepatectomy led to proliferation of the trans- samples of standard liver DNA mixtures. Individual samples planted hepatocytes, resulting in a slightly higher en- ranging from 20 to 0.01% male liver DNA are plotted as cycle graftment of donor cells (8,18). number (C ) against log of the concentration. Linear regression analysis is shown for each gene target. Fas, a type II TNF family receptor, induces intracel- 358 FUJINO ET AL. Figure 5. Histological findings and serum transaminase activities in the Jo2-treated mice. (A) AST concentrations were increased after 24 h when injected Jo2 at a dose of 0.15 mg/kg at the first time and 0.3 mg/kg at the second time. However, there was no dramatic change in AST when injecting the same dose as the first time, indicating the mice appeared to acquire resistance against the antibody. (B) Histological study of the liver from Jo2-treated mice showed a severe hepatocyte vacuolation, multifocal hepatocyte death, and increased number of inflammatory cells (e, f), while there was slight damage after the second injection of the Jo2 antibody at the same dose as the first time in normal mice (g), but not in lpr mice (h). The observation in TUNEL staining sample is consistent with H&E findings in a different manner (a–d). REPOPULATION OF FAS-RESISTANT HEPATOCYTE TRANSPLANT 359 Table 1. Hepatocyte Transplantation in Jo2-Treated Animals Ratio of Transplanted Cell DNA Treatment % Mean ± SE Mean ± SE Jo2 (anti-Fas antibody) 1 month E-1-1 3.3 E-1-2 1.3 E-1-3 1.6 1.5 ± 0.5 E-1-4 0.4 ] E-1-5 1.1 2 months E-2-1 2.8 E-2-2 6.9 E-2-3 3.6 3.1 ± 1.1 E-2-4 1.0 ] E-2-5 1.1 3 months E-3-1 1.2 2.9 ± 0.9 E-3-2 1.4 ] E-3-3 4.2 2.7 ± 0.7 E-3-4 4.4 ] E-3-5 2.2 Saline (control) 1 month C-1-1 0.2 C-1-2 0.1 C-1-3 0.1 ] 0.1 ± 0.03 C-1-4 0.03 C-1-5 0.03 2 months C-2-1 0.1 C-2-2 0.1 C-2-3 0.1 0.1 ± 0.01 C-2-4 0.1 ] C-2-5 0.1 3 months C-3-1 0.1 0.1 ± 0.03 C-3-2 0.1 ] C-3-3 0.1 0.1 ± 0.3 C-3-4 0.1 ] C-3-5 0.3 lular apoptotic signals through the caspase cascade. In- liver that contains approximately 1 × 10 hepatocytes. jection of an agonistic anti-Fas antibody (Jo2) via the The repopulation in the experimental group resulted tail vein into mice resulted in hepatitis accompanied by from the specific resistance of lpr hepatocytes to Fas- Fas-mediated apoptosis. In our experimental setting, we mediated apoptosis. In the control group, as expected, induced Fas-mediated hepatocyte apoptosis in vivo to the livers of mice transplanted with lpr hepatocytes but stimulate hepatocellular regeneration. A previous study without Jo2 treatments were not repopulated by these indicated that a sublethal dose of Jo2 kills approximately cells (Table 1). This result is consistent with the absence one third of the resident hepatocytes. We assumed that of repopulation of FAH−/− mice livers transplanted with both apoptosis-sensitive resident hepatocytes and apop- FAH+/+ hepatocytes and treated with NTBC (13). In tosis-resistant lpr hepatocytes participate in the regenera- this study, the administration of Jo2 after transplantation 6 6 tive process. We injected 1 × 10 lpr hepatocytes into a of 1 × 10 lpr hepatocytes resulted in repopulation of the recipient spleen, where we expected approximately 20% donor hepatocytes at 6.9% in the recipient livers. This (2 × 10 cells) of the cells to redistribute and survive in result was 70-fold higher than that obtained with infu- the liver (14,15). As expected, the number of repopulat- sion of the same number of hepatocytes with control ing lpr hepatocytes after three treatments of Jo2 was saline. 5 3 5 2 × 10 × (3/2) = 6.75 × 10 . These results correspond to Our results yielded evidence that lpr-derived hepato- the optimal percentage of 0.68% for a normal mouse cytes would contribute to repopulation in Fas-positive 360 FUJINO ET AL. assessed on an automate sequenator. A highly sensitive and reproducible method with a rapid turnaround time is required, especially in a clinical setting. We demonstrated in this study that there was no de- tectable signal when genomic male DNA was tested in the control female hepatocytes, thus indicating the speci- ficity of this primer pair and the fluorescent TaqMan probe set for the sry gene. In addition to the favorable comparison with the technique of quantitative competi- tive PCR, this new real-time PCR approach has several advantages over the myriad of other techniques that have been used to assess gene transfer or gene correc- tion. Furthermore, the potential extension of this meth- Figure 6. Effects of Jo2-induced regeneration of donor hepa- odology to RT-PCR cannot be overestimated. Such an tocytes transplantation. Mice were given Jo2 or normal saline accurate and sensitive method has the potential to mea- 1 day after hepatocyte transplantation. Mice were sacrificed sure any biologic system. 1–3 months after transplantation. The data show the percent- age of mouse sry gene in five recipients. The data are pre- In conclusion, we demonstrated that transplanted lpr sented as mean ± SE. hepatocytes repopulated significantly in a normal liver after selective destruction of the host hepatocytes by three administrations of the agonistic anti-Fas antibody hepatocytes of a normal liver when treated three times at sublethal doses. Estimation of the sry gene by quanti- with anti-Fas antibody at sublethal doses. Similar ap- tative real-time PCR is useful for defining the repopula- proaches using transgenic mice with bcl-2 genes or bcl- tive efficiency of transplanted male hepatocytes in a fe- 2-transduced hepatocytes using an adeno-associated vi- male liver. rus or lentivirus could allow reconstitution of a large ACKNOWLEDGMENTS: We thank Drs. H. Kimura and S. number of these hepatocytes in an anti-Fas antibody- Enosawa for their comments and useful suggestions. This study was supported by a Grant for Organized Research Com- treated recipient (7,11,16). 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Journal

Cell TransplantationSAGE

Published: Jul 7, 2017

Keywords: Fas; Hepatocyte transplantation; Real-time quantitative PCR; sry gene

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