- Email: [email protected]
Hematopoietic Stem Cell Mobilization After Rat Partial Orthotopic Liver Transplantation
Hematopoietic Stem Cell Mobilization After Rat Partial Orthotopic Liver Transplantation F. Liu, X.B. Pan, G.D. Chen, D. Jiang, X. Cong, R. Fei, H.S. Chen, and L. Wei ABSTRACT Background. On the basis of the recently recognized potential of hematopoietic stem cells (HSC) to give rise to hepatocytes, we investigated the possibility that HSC could be mobilized and home to the injured liver promoting tissue repair after 50% partial orthotopic liver transplantation (PLTx) in the rat. Methods. Using sex-mismatched (female to male) syngeneic SD rats, we performed 50% PLTx or whole orthotopic liver transplantation (WLTx) versus 50% partial hepatectomy (PHx) and sham operation (O). Elements with stem cell markers were detected in peripheral blood (PB) and in the liver. Liver injury and regeneration were estimated. The sex-determining region for Y chromosome gene (SRY) was used to define cell origin by in situ hybridization in liver sections. Results. Comparison of WLTx and PHx groups showed a lower survival rate (50%), in the PLTx group were (P ⬍ .05). Further, the liver injury was more serious and the levels of serum biochemical parameters were higher. Compared with PHx groups, on days 3 and 5 postoperatively, the mitosis index and the expression of PCNA were lower among the PLTx groups. Compared with WLTx and sham operation groups, 2m-/Thy-1.1⫹, CD34⫹ cells in PB in PLTx groups and PHx were increased on day 1 postoperatively and decreased on the following days. Compared with PHx groups, 2m-/Thy-1.1⫹, CD34⫹ cells were higher in PLTx. The CD34-, c-kit-, and Thy-1.1-positive cells detected in portal tract areas peaked during 3 to 5 days postoperatively in PLTx. Few SRY⫹ cells were detected in PLTx liver grafts. Conclusions. 2m-/Thy-1.1⫹ and CD34⫹ stem cells mobilized after PLTx and PHx may be related to the reduced-size liver. Few HSC are involved in liver regeneration in PLTx.
T
HE GROWING ORGAN shortage for transplantation has led to an increased use of partial orthotopic liver transplantation (PLTx). The major concern in this endeavor is the adequacy of the size of the graft.1,2 A small-for-size graft may not only be functionally inadequate for the recipient but also will be prone to sustain rejection and ischemic injuries resulting in inadequate regeneration, hepatic insufficiency, or overt liver failure. Therefore, improved liver regeneration capacity may be of critical importance in PLTx. Recent studies have suggested that hematopoietic stem cells (HSCs) can differentiate into nonhematopoietic lineages in vivo, including skeletal3 and cardiac myoblasts4; vascular endothelium5; epithelial cells of lung, gut, and skin6; and neuroectodermal cells.7,8 It is not yet clear whether the underlying cellular mechanism of this apparent
plasticity is transdifferentiation of HSCs,9 –11 or cell fusion between HSCs and target cells.12,13 There are several reports on the ability of HSCs to generate hepatocytes From the Hepatology Institute (F.L., X.B.P., D.J., X.C., R.F., H.S.C., L.W.), Department of Gastroenterology (G.D.C.), Peking University People’s Hospital, Beijing, People’s Republic of China. Supported by the National High Technology Research and Development Program of China (863 Program) (2001AA216031) from Administration of Science and Technology of China, Chinese Basic Research Foundation (973) (2005CB522902) and Ministry of Education of P.R. China (20040001133). Address reprint requests to Wei Lai, MD, Professor, Hepatology Institute, Peking University, People’s Hospital, No. 11 Xizhimen South Street, Beijing, 100044, P.R. China. E-mail: weelai@ 163.com
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.02.121
Transplantation Proceedings, 38, 1603–1609 (2006)
1603
1604
under tissue stress in mice and humans.14 –18 Therefore, it has been proposed that HSC migration into sites of injury may be a repair mechanism for damaged tissues.19 The present study was designed to explore the possibility that auto-HSCs could be mobilized and home to the injured liver promoting tissue repair after PLTx. MATERIALS AND METHODS Animals Inbred Sprague Dawley (SD) rats (Academy of Military Medical Science), weighing 250 to 300 g were housed with free activity and access to water and chow. The constant environmental conditions had a 12-hour light/dark cycle. The rats were fasted 12 hours before operation. All animal experiments were conducted in accordance with the National Research Council’s guide for the care and use of laboratory animals. Animal survival was checked every 8 hours. At the time of death, a necropsy was performed to detect technical complications, and the graft was removed for histological examination.
Experimental Procedure Orthotopic liver transplantation was performed using the two-cuff method.20 Animals were divided into four groups: group 1, female SD to male SD 50% PLTx group, donor left hemihepatectomy including left lateral, left median, and anterior and posterior caudate lobes, was followed by harvest of the liver; the reduced-size graft then was implanted orthotopically into the recipient. Group 2 was female SD to male SD whole orthotopic liver transplantation (WLTx) group. Group 3 was the male 50% partial hepatectomy (PHx) group with half of the liver resected the same as donors of PLTx. Group 4 was sham-operated, consisting of a laparotomy with mobilization of the liver. Six animals were killed at 1, 3, 5, 7, and 14 days after transplantation. At the time of sacrifice, blood was obtained from the inferior vena cava to estimate serum alanine transferase (ALT) and aspartate transferase (AST) activities. Portions of the liver fixed in 10% neutral buffered formalin for 18 to 24 hours were processed for light microscopy. The remainder of the liver was frozen in liquid nitrogen for storage at ⫺80°C.
Survival Study Ten rats in PLTx, WLTx, and PHx groups were used for the survival study. Rat deaths within 48 hours of operation were considered to be technical failures and excluded.
LIU, PAN, CHEN ET AL bers were determined by serial FACS analysis between days 1 and 14 after operation. The mononuclear cells were isolated by Ficoll-Paque (Amersham Pharmacia Biotech, Freiburg, Germany) density gradient centrifugation of heparinized ethylenediamine-tetraacetic acid (EDTA) blood samples. The cells were suspended in FACS buffer (phosphate-buffered saline supplemented with 5% fetal borne serum and containing 0.05% NaN3) at a concentration of about 1,000,000 cells/mL. The cells were washed by centrifugation in three volumes of FACS buffer and resuspended in 50 L FACS buffer containing primary antibody for 40 minutes at 4°C. For the isotype control, nonspecific rat immunoglobulin G (IgG) was substituted for primary antibody. For antibodies that required a second antibody for detection, the cell pellet was incubated under the same conditions for 20 minutes with anti-rabbit IgG labeled with fluorescein isothiocyanate (FITC). Cells were washed three times with FACS buffer and fixed in PBS/4% paraformaldehyde for 30 minutes on ice for analysis. The primary antibodies were FITC-conjugated mouse anti-rat 2m monoclonal antibody, phycoerythrin-conjugated mouse anti-rat Thy1.1 (BD Pharmingen), FITC-conjugated mouse anti-rat CD45 (Harlan Lab), PEconjugated rabbit anti-rat CD34, rabbit anti-rat C-kit, and rabbit anti-rat flt-2/3 (Santa Cruz). The secondary antibodies were FITCconjugated goat anti-rabbit IgG (BD Pharmingen).
Immunohistochemistry of CD34, Thy-1.1, and c-kit Antigen Immunohistochemical detection was performed on serial frozen sections after 10-minute fixation in cold acetone; 6 m-thick sections were incubated in a 1:10 dilution of anti-rat polyclonal CD34 antibody, 1:8 anti-rat monoclonal Thy1.1 antibody, or 1:10 anti-rat C-kit antibody (Santa Cruz Biotechnology). The immunohistochemistry was performed using a commercial kit (Zymed LAB-SA System, Zymed Laboratories). Peroxidase activity was determined by diaminobenzidine staining. The sections were counterstained with hematoxylin.
ISH of the Y Chromosomes The probe derived from the sequence of SRY was a 104 bp pair polymerase chain reaction product amplified from male genomic DNA using PCR DIG Probe Synthesis Kit (Boehringer Mannheim, Germany). The primer sequences were F, CAT CGA AGG GTT AAA GTG CCA and R, ATA GTG TGT AGG TTG TTG TCC.
Evaluation of Injury and Regeneration For histological analysis of liver grafts, paraffin-embedded 6 mthick sections were stained with hematoxylin and eosin (H&E). To evaluate regeneration, mitotic activity in the livers was evaluated in 6 m-thick sections stained with H&E. Paraffin sections were stained with antibodies to PCNA (Santa Cruz Biotechnology, Santa Cruz, Calif, USA). The immunohistochemistry was performed using a commercial kit (Zymed LAB-SA System, Zymed Laboratories, South San Francisco, Calif, USA). The hepatic labeling index was calculated as the number of PCNA-labeled nuclei per 1000 hepatocyte nuclei. In addition, mitotic activity was also evaluated by counting 1000 hepatocytes in each liver.
Mobilization of HSC by FACS To assess whether PLTx per se resulted in an increase in peripheral HSC numbers, 2m-/Thy-1.1⫹, CD34⫹, c-kit, and flt-2/3 cell num-
Fig 1. Survival rates. The survival of rats in the PLTx group was significantly lower compared with those of WLTx and PHx groups.
HEMATOPOIETIC STEM CELL MOBILIZATION
1605
Fig 2. Histology of the grafts on day 14 after PLTx. (A) H&E-stained liver section from PLTx group graft with focal and small areas of hepatocytes necrosis. (B) Liver section from PHx group. (C) Liver sections from WLTx group. Note the normal histology of liver lobule. (Original magnification [A,C] ⫻200, [B] ⫻300.) The polymerase chain reaction amplification was performed under the following conditions in a thermal cycler (Perkin-Elmer/Cetus). After denaturation at 94°C for 5 minutes, 35 cycles of amplification were performed; each step consisted of denaturation at 94°C for 30 seconds, primer annealing at 56°C for 30 seconds, and chain elongation at 72°C for 45 seconds, followed by a final elongation step at 72°C for 7 minutes. The probe was purified using the QIAquick Gel Purification Kit (QIAGEN), according to the manufacturer’s instructions. The in situ hybridization reaction was performed on paraffin 6 m tissue sections. After removal of paraffin, sections were treated by proteolytic digestion for 30 minutes at 37°C with 10 g/mL proteinase K (Sigma, St Louis, Mo, USA) dissolved in 50 mmol EDTA and 0.1 mol/L Tris-HCl, followed by two rinses of 5 minutes each in 0.01 mol/L PBS. After washing, the specimens were prehybridized for 30 minutes with buffer containing 10% dextran sulfate, 5 ⫻ standard saline citrate (SSC), 5 ⫻ Denhardt’s Solution (Roche), 100 g/mL salmon sperm DNA, and 50% deionized formamide (Sigma). Residual prehybridization buffer was removed from the tissue sections and the appropriate digoxygenin probes, diluted in prehybridization buffer, were applied to the sections. The sections were covered with siliconecoated coverslips (Sigma), denatured at 95°C for 5 minutes on a heat plate, and hybridized in humidified atmosphere at 37°C overnight. Upon completion of the hybridization reaction, the coverslips were removed and specimens washed three times for 10 minutes each in 50% formamide in 2 ⫻ SSC solution at 42°C, followed by two washes in 2 ⫻ SSC for 5 minutes each. Five percent borne serum albumin (BSA) blocking reagent was then applied for 30 minutes and drained away, then alkaline phosphatase-conjugated sheep anti-digoxygenin antibody (Roche Molecular Biochemicals), diluted 1:10 in 5% BSA, was applied for 1 hour at 37°C. Sections were rinsed three times for 5
minutes each in 4 ⫻ SSC/0.01% Tween solution at 42°C, and then reacted with nitroblue tetrazolium/5-bromocresyl-3-indolylphosphate chromogen (Roche Molecular Biochemicals) for 30 minutes. Sections were rinsed again and counterstained with 0.1% nuclear fast red and analyzed under a light microscope. Some tissue sections were processed without probe to serve as negative controls. SRY⫹ cells were evaluated by counting positive cells per 1000 hepatocytes in each liver.
Statistical Analysis Continuous variables were expressed as mean values ⫾ SD. Differences of continuous parameters among groups were subjected to one-way analysis of variance followed by Scheffe’s post hoc test. A value of P ⬍ .05 was considered significant. Animal survival was evaluated using Kaplan-Meyer plots. The data were analyzed using SPSS software version 11.0 (SPSS Inc, Chicago, Ill, USA).
RESULTS Survival Rate Was Low in PLTx
Graft survivals are shown in Fig 1. In the PLTx group, the rate of survival at 7 days was 5 of 10. In contrast, recipients in the WLTx and PHx groups survived indefinitely. Liver Injury Was Serious in PLTx
On days 7 and 14 postoperatively, livers from the PHx group maintained a normal architecture. Livers from the WLTx group rats demonstrated only scattered hepatocyte necrosis, maintaining a rather normal architecture. In contrast, livers from the PLTx group showed hepatocellular necrosis around the portal tract areas (Fig 2A to 2C).
Fig 3. Serum ALT and AST activity. (A) Serum ALT and (B) AST activities after operation (mean ⫾ SD) (n ⫽ 6). Vertical bars represent the SD from the mean. Statistical significance. #P ⬍ .05 for PLTx versus PHx and WLTx livers at the different time points. *P ⬍ .05 for PLTx versus PHx and WLTx groups at the same time points.
1606
LIU, PAN, CHEN ET AL
Fig 4. Hepatocyte-proliferative activity of liver after operation. Percentage of (A) mitoses and (B) PCNA index (mean ⫾ SD). # P ⬍ .05 for PLTx versus PHx and WLTx at the different time points. *P ⬍ .05 for PLTx liver versus PHx at the same time points.
Serum AST and ALT levels increased on day 1 and decreased on the following days in the PLTx, PHx, and WLTx groups. The PLTx values were significantly higher than those in the PHx or WLTx groups at various times (P ⬍ .05); (Fig 3). These findings suggested that liver injury was more serious in the PLTx group. The Capacity of Liver Regeneration was Lower in PLTx
The percentages of liver regeneration parameters are shown in Fig 4. Compared with the PHx group, the livers in the PLTx group showed significantly lower regeneration rates on day 1 and day 3, and higher regeneration rates on days 7 and 14. The maximum PCNA index of hepatocytes was 65.83% ⫾ 13.00% for PHx rats, 47.00% ⫾ 6.81% for PLTx rats (P ⬍ .05). The cells proliferating at 3 days after PLTx were predominantly in the periportal and midzonal region of the liver (Fig 5A to 5C). These data suggested that regeneration in a partial liver graft was decreased and delayed compared with PHx. HSC Mobilization and Homing to Liver Graft After PLTx
The changes in 2m-/Thy-1.1⫹ and CD34⫹ cells in PB are shown in Fig 6. Compared with WLTx and sham groups, 2m-/Thy-1.1⫹ and CD34⫹ cells in peripheral blood (PB) in PLTx and PHx groups increased on day 1 postoperatively and decreased on the following days. Compared with PLTx group, 2m-/Thy-1.1⫹ and CD34⫹ cells were higher than those in PHx on day 1 (P ⬍ .05). The number of c-kit⫹ and Flt-2/3⫹ cells in PB displayed no change at various times.
Fig 5. Immunohistochemical staining of PCNA-positive nuclei. (A) PLTx group showed lower positive cells number than (B) PHx group. (C) Liver in WLTx showed few positive cells. Brown-stained hepatocytes represent positive staining, indicating hepatocytes in S through M phases of the cell cycle. (Original magnification ⫻200.)
We assessed stem cell migration into liver grafts using sections stained with CD34, c-kit, and Thy-1.1. Staining by all three antibodies on serial sections showed similar patterns: with all staining was located in the periportal region spreading outward, but CD34⫹ cells were also c-kit⫹ and thy1.1⫹ or not. At higher magnification, the reactivity of the antibodies was primarily on infiltrated monocytes, or what other investigators have termed “transitional cells.” Also, in PLTx, a few sinusoidal lining cells and bile ductules were positive for c-kit, whereas in the normal liver, they were negative, as in Fig 7E and Fig 7F with two different staining methods. SRY⫹ Cells in Liver Grafts
In the male rat (positive control), SRY positivity was observed in the majority of hepatocytes (Fig 8A). Contaminating hematopoetic cells, preferentially arranged periportally, were easily distinguished by morphology and excluded from counting. In the PLTx group, few SRY⫹ hepatocytes were observed (Fig 8C). DISCUSSION
It has been reported that bone marrow– derived stem cells can give rise to hepatocytes, oval cells, and bile duct cells.15,17 Therefore, it has been proposed that HSC, can migrate into sites of injury undergoing tissue-specific differentiation that promotes structural and functional repair. To test whether HSC could be mobilized and
HEMATOPOIETIC STEM CELL MOBILIZATION
1607
Fig 6. The change of 2m-/Thy1.1⫹ and CD34⫹ cells in PB in four groups by FACS. The change of (A) 2m-/Thy-1.1⫹ and (B) CD34⫹ cells. *P ⬍ .05 for PLTx and PHx versus WLTx livers at the same time points, respectively.
naturally migrate into the liver after hepatic injury and have the capacity to promote liver repair, we used a sex-mismatched rat PLTx model. In this model donorderived hepatocytes are detected using the Y chromosome as a marker. First, we evaluated the injury and regeneration of the
liver. Compared with PHx and WLTx, the PLTx cases showed worse histology of the liver grafts, lower survival rates were and higher levels of AST and ALT. These results indicated that liver injury in PLTx was more severe. The mitosis index and PCNA in PLTx were lower than those in PHx, which suggested that the capacity of
Fig 7. Immunohistochemical staining of CD34⫹ and c-kit⫹ cells on day 3 after transplantation. The brown-stained CD34⫹ and c-kit⫹ cells were mainly in the portal tract areas. More CD34⫹ cells were found in (A) PLTx than (B) WLTx and more c-kit⫹ cells were found in (C) PLTx than (D) WLTx. The (E) blue-purple and (F) red-stained c-kit⫹ was showed on duct epithelium. (Original magnification ⫻400.)
1608
LIU, PAN, CHEN ET AL
Fig 8. ISH for SRY⫹ cells (bluepurple dot, arrow) in the liver. (A) A male liver specimen showed that there were Y chromosome signals (indicated by arrows) in hepatocytes. (B) A female liver specimen showed that there were no Y chromosome signals in hepatocytes. (C) A femalemale isograft specimen in the PLTx group showed that there were few Y chromosome signals (indicated by arrows) in hepatocytes. (Original magnification [A,C] ⫻400, [B] ⫻300.)
liver graft regeneration was decreased. In PLTx, liver injury was related not only to loss of liver mass but also to reperfusion and early immune responses,21 which may explain the difference between liver injury and regeneration in PLTx versus PHx. Then, we studied the role of HSC mobilization focusing on 2m-/Thy-1.1⫹, CD34⫹, c-kit⫹, and flt-2/3⫹ HSCs, which are precursors of blood and endothelial cells. We observed that CD34⫹ and 2m-/Thy-1.1⫹ cells in PB were increased on day 1 after operation in PLTx and PHx but not in WLTx and sham groups, which indicated that HSC were mobilized, probably because of the loss of liver mass. Howerver, we found CD34⫹ and 2m-/Thy-1.1⫹ cells in PLTx to show higher levels than those in PHx CD34⫹ and Thy-1.1⫹ cells were increased in partial liver grafts, but not in PHx or WLTx. These results indicated that more CD34⫹ and Thy-1.1 cells were mobilized and homed to the liver graft after PLTx, which may depend on a more serious liver graft injury and an increased immune response22 in rat PLTx. In addition, we found c-kit⫹ cells, partly expressed on duct epithelium, increased in the liver graft but not in PB. The c-kit protein (CD117) has been identified as the natural receptor for the cytokine stem cell factor, representing a marker of both HSC and oval cells.23 It has been suggested that activated endogenous liver oval cells may be involved in regeneration. HSC contribution to liver regeneration was evaluated by ISH for SRY in paraffin sections after sex-mismatched transplantation. SRY is a transcriptional activator and although its use as a marker could generate concerns of false negativity in quiescent cells outside the male reproductive system, we provided evidence for SRY detection in resting cells, because the vast majority of cells in male control livers were SRY⫹. We found few SRY⫹ cells in PLTx, which suggested few exogenous cells were involved in regeneration. Analysis of liver regeneration and SRY⫹ cells suggested liver restoration was mainly endogenous, with less support from HSCs, because the majority of proliferating cells were of host, rather than HSC, origin. HSCs, mobilized after PLTx and PHx, may be a normal
phenomenon to respond to stress signals from the injured liver. Studies24 have shown that the chemokine SDF-1 and its receptor CXCR4 participate in the mobilization of HSCs from bone marrow and in the migration of HSCs to the injured liver. However, liver regeneration mainly focused on endogenous hepatic restoration programs. The precise impact of HSC mobilization and migration on partial liver grafts remains unknown.
REFERENCES 1. Kiuchi T, Kasahara M, Uryuhara K, et al: Impact of graft size mismatching on graft prognosis in liver transplantation from living donors. Transplantation 67:321, 1999 2. Lo CM, Fan ST, Liu CL, et al: Minimum graft size for successful living donor liver transplantation. Transplantation 68: 1112, 1999 3. Ferrari G, Cusella-De Angelis G, Coletta M, et al: Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279:1528, 1998 4. Krause DS, Theise ND, Collector MI, et al: Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105:369, 2001 5. Lin Y, Weisdorf DJ, Solovey A, et al: Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 105:71, 2000 6. Asahara T, Murohara T, Sullivan A, et al: Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964, 1997 7. Brazelton TR, Rossi FM, Keshet Gl, et al: From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290:1775, 2000 8. Mezey E, Chandross KJ, Harta G, et al: Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 290:1779, 2000 9. Jang YY, Collector MI, Baylin SB, et al: Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 6:532, 2004 10. Harris RG, Herzog EL, Bruscia EM, et al: Lack of a fusion requirement for development of bone marrow-derived epithelia. Science 305:90, 2004 11. Ianus A, Holz GG, Theise ND, et al: In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 111:843, 2003 12. Wang X, Willenbring H, Akkari Y, et al: Cell fusion is the principal source of bone marrow-derived hepatocytes. Nature 422:897, 2003
HEMATOPOIETIC STEM CELL MOBILIZATION 13. Vassilopoulos G, Wang PR, Russell DW: Transplanted bone marrow regenerates liver by cell fusion. Nature 422:901, 2003 14. Petersen BE, Bowen WC, Patrene KD, et al: Bone marrow as a potential source of hepatic oval cells. Science 284:1168, 1999 15. Theise ND, Badve S, Saxena R, et al: Derivation of hepatocytes from bone marrow cells of mice after radiation-induced myeloablation. Hepatology 31:235, 2000 16. Theise ND, Nimmakayalu M, Gardner R, et al: Liver from bone marrow in humans. Hepatology 32:11, 2000 17. Korbling M, Katz RL, Khanna A, et al: Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med 346:738, 2002 18. Alison MR, Poulsom R, Jeffery R, et al: Hepatocytes from non-hepatic adult stem cells. Nature 406:257, 2000 19. Wright DE, Bowman EP, Wagers AJ, et al: Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. J Exp Med 195:1145, 2002
1609 20. Kamada N, Calne RY: A surgical experience with five hundred and thirty liver transplantations in the rat. Surgery 93:64, 1983 21. Dirsch O, Dahmen U, Gu YL, et al: Influence of cold ischemia on liver regeneration after partial liver transplantation. Transplant Proc 34:2303, 2002 22. Omura T, Nakagawa T, Randall HB, et al: Increased immune responses to regenerating partial liver grafts in the rat. J Surg Res 70:34, 1997 23. Matsusaka S, Tsujimura T, Toyosaka A, et al: Role of c-kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminofluorene/partial hepatectomy model. Hepatology 29: 670, 1999 24. Kollet O, Shivtiel S, Chen YQ, et al: HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34⫹ stem cell recruitment to the liver. J Clin Invest 112:160, 2003
T
HE GROWING ORGAN shortage for transplantation has led to an increased use of partial orthotopic liver transplantation (PLTx). The major concern in this endeavor is the adequacy of the size of the graft.1,2 A small-for-size graft may not only be functionally inadequate for the recipient but also will be prone to sustain rejection and ischemic injuries resulting in inadequate regeneration, hepatic insufficiency, or overt liver failure. Therefore, improved liver regeneration capacity may be of critical importance in PLTx. Recent studies have suggested that hematopoietic stem cells (HSCs) can differentiate into nonhematopoietic lineages in vivo, including skeletal3 and cardiac myoblasts4; vascular endothelium5; epithelial cells of lung, gut, and skin6; and neuroectodermal cells.7,8 It is not yet clear whether the underlying cellular mechanism of this apparent
plasticity is transdifferentiation of HSCs,9 –11 or cell fusion between HSCs and target cells.12,13 There are several reports on the ability of HSCs to generate hepatocytes From the Hepatology Institute (F.L., X.B.P., D.J., X.C., R.F., H.S.C., L.W.), Department of Gastroenterology (G.D.C.), Peking University People’s Hospital, Beijing, People’s Republic of China. Supported by the National High Technology Research and Development Program of China (863 Program) (2001AA216031) from Administration of Science and Technology of China, Chinese Basic Research Foundation (973) (2005CB522902) and Ministry of Education of P.R. China (20040001133). Address reprint requests to Wei Lai, MD, Professor, Hepatology Institute, Peking University, People’s Hospital, No. 11 Xizhimen South Street, Beijing, 100044, P.R. China. E-mail: weelai@ 163.com
© 2006 by Elsevier Inc. All rights reserved. 360 Park Avenue South, New York, NY 10010-1710
0041-1345/06/$–see front matter doi:10.1016/j.transproceed.2006.02.121
Transplantation Proceedings, 38, 1603–1609 (2006)
1603
1604
under tissue stress in mice and humans.14 –18 Therefore, it has been proposed that HSC migration into sites of injury may be a repair mechanism for damaged tissues.19 The present study was designed to explore the possibility that auto-HSCs could be mobilized and home to the injured liver promoting tissue repair after PLTx. MATERIALS AND METHODS Animals Inbred Sprague Dawley (SD) rats (Academy of Military Medical Science), weighing 250 to 300 g were housed with free activity and access to water and chow. The constant environmental conditions had a 12-hour light/dark cycle. The rats were fasted 12 hours before operation. All animal experiments were conducted in accordance with the National Research Council’s guide for the care and use of laboratory animals. Animal survival was checked every 8 hours. At the time of death, a necropsy was performed to detect technical complications, and the graft was removed for histological examination.
Experimental Procedure Orthotopic liver transplantation was performed using the two-cuff method.20 Animals were divided into four groups: group 1, female SD to male SD 50% PLTx group, donor left hemihepatectomy including left lateral, left median, and anterior and posterior caudate lobes, was followed by harvest of the liver; the reduced-size graft then was implanted orthotopically into the recipient. Group 2 was female SD to male SD whole orthotopic liver transplantation (WLTx) group. Group 3 was the male 50% partial hepatectomy (PHx) group with half of the liver resected the same as donors of PLTx. Group 4 was sham-operated, consisting of a laparotomy with mobilization of the liver. Six animals were killed at 1, 3, 5, 7, and 14 days after transplantation. At the time of sacrifice, blood was obtained from the inferior vena cava to estimate serum alanine transferase (ALT) and aspartate transferase (AST) activities. Portions of the liver fixed in 10% neutral buffered formalin for 18 to 24 hours were processed for light microscopy. The remainder of the liver was frozen in liquid nitrogen for storage at ⫺80°C.
Survival Study Ten rats in PLTx, WLTx, and PHx groups were used for the survival study. Rat deaths within 48 hours of operation were considered to be technical failures and excluded.
LIU, PAN, CHEN ET AL bers were determined by serial FACS analysis between days 1 and 14 after operation. The mononuclear cells were isolated by Ficoll-Paque (Amersham Pharmacia Biotech, Freiburg, Germany) density gradient centrifugation of heparinized ethylenediamine-tetraacetic acid (EDTA) blood samples. The cells were suspended in FACS buffer (phosphate-buffered saline supplemented with 5% fetal borne serum and containing 0.05% NaN3) at a concentration of about 1,000,000 cells/mL. The cells were washed by centrifugation in three volumes of FACS buffer and resuspended in 50 L FACS buffer containing primary antibody for 40 minutes at 4°C. For the isotype control, nonspecific rat immunoglobulin G (IgG) was substituted for primary antibody. For antibodies that required a second antibody for detection, the cell pellet was incubated under the same conditions for 20 minutes with anti-rabbit IgG labeled with fluorescein isothiocyanate (FITC). Cells were washed three times with FACS buffer and fixed in PBS/4% paraformaldehyde for 30 minutes on ice for analysis. The primary antibodies were FITC-conjugated mouse anti-rat 2m monoclonal antibody, phycoerythrin-conjugated mouse anti-rat Thy1.1 (BD Pharmingen), FITC-conjugated mouse anti-rat CD45 (Harlan Lab), PEconjugated rabbit anti-rat CD34, rabbit anti-rat C-kit, and rabbit anti-rat flt-2/3 (Santa Cruz). The secondary antibodies were FITCconjugated goat anti-rabbit IgG (BD Pharmingen).
Immunohistochemistry of CD34, Thy-1.1, and c-kit Antigen Immunohistochemical detection was performed on serial frozen sections after 10-minute fixation in cold acetone; 6 m-thick sections were incubated in a 1:10 dilution of anti-rat polyclonal CD34 antibody, 1:8 anti-rat monoclonal Thy1.1 antibody, or 1:10 anti-rat C-kit antibody (Santa Cruz Biotechnology). The immunohistochemistry was performed using a commercial kit (Zymed LAB-SA System, Zymed Laboratories). Peroxidase activity was determined by diaminobenzidine staining. The sections were counterstained with hematoxylin.
ISH of the Y Chromosomes The probe derived from the sequence of SRY was a 104 bp pair polymerase chain reaction product amplified from male genomic DNA using PCR DIG Probe Synthesis Kit (Boehringer Mannheim, Germany). The primer sequences were F, CAT CGA AGG GTT AAA GTG CCA and R, ATA GTG TGT AGG TTG TTG TCC.
Evaluation of Injury and Regeneration For histological analysis of liver grafts, paraffin-embedded 6 mthick sections were stained with hematoxylin and eosin (H&E). To evaluate regeneration, mitotic activity in the livers was evaluated in 6 m-thick sections stained with H&E. Paraffin sections were stained with antibodies to PCNA (Santa Cruz Biotechnology, Santa Cruz, Calif, USA). The immunohistochemistry was performed using a commercial kit (Zymed LAB-SA System, Zymed Laboratories, South San Francisco, Calif, USA). The hepatic labeling index was calculated as the number of PCNA-labeled nuclei per 1000 hepatocyte nuclei. In addition, mitotic activity was also evaluated by counting 1000 hepatocytes in each liver.
Mobilization of HSC by FACS To assess whether PLTx per se resulted in an increase in peripheral HSC numbers, 2m-/Thy-1.1⫹, CD34⫹, c-kit, and flt-2/3 cell num-
Fig 1. Survival rates. The survival of rats in the PLTx group was significantly lower compared with those of WLTx and PHx groups.
HEMATOPOIETIC STEM CELL MOBILIZATION
1605
Fig 2. Histology of the grafts on day 14 after PLTx. (A) H&E-stained liver section from PLTx group graft with focal and small areas of hepatocytes necrosis. (B) Liver section from PHx group. (C) Liver sections from WLTx group. Note the normal histology of liver lobule. (Original magnification [A,C] ⫻200, [B] ⫻300.) The polymerase chain reaction amplification was performed under the following conditions in a thermal cycler (Perkin-Elmer/Cetus). After denaturation at 94°C for 5 minutes, 35 cycles of amplification were performed; each step consisted of denaturation at 94°C for 30 seconds, primer annealing at 56°C for 30 seconds, and chain elongation at 72°C for 45 seconds, followed by a final elongation step at 72°C for 7 minutes. The probe was purified using the QIAquick Gel Purification Kit (QIAGEN), according to the manufacturer’s instructions. The in situ hybridization reaction was performed on paraffin 6 m tissue sections. After removal of paraffin, sections were treated by proteolytic digestion for 30 minutes at 37°C with 10 g/mL proteinase K (Sigma, St Louis, Mo, USA) dissolved in 50 mmol EDTA and 0.1 mol/L Tris-HCl, followed by two rinses of 5 minutes each in 0.01 mol/L PBS. After washing, the specimens were prehybridized for 30 minutes with buffer containing 10% dextran sulfate, 5 ⫻ standard saline citrate (SSC), 5 ⫻ Denhardt’s Solution (Roche), 100 g/mL salmon sperm DNA, and 50% deionized formamide (Sigma). Residual prehybridization buffer was removed from the tissue sections and the appropriate digoxygenin probes, diluted in prehybridization buffer, were applied to the sections. The sections were covered with siliconecoated coverslips (Sigma), denatured at 95°C for 5 minutes on a heat plate, and hybridized in humidified atmosphere at 37°C overnight. Upon completion of the hybridization reaction, the coverslips were removed and specimens washed three times for 10 minutes each in 50% formamide in 2 ⫻ SSC solution at 42°C, followed by two washes in 2 ⫻ SSC for 5 minutes each. Five percent borne serum albumin (BSA) blocking reagent was then applied for 30 minutes and drained away, then alkaline phosphatase-conjugated sheep anti-digoxygenin antibody (Roche Molecular Biochemicals), diluted 1:10 in 5% BSA, was applied for 1 hour at 37°C. Sections were rinsed three times for 5
minutes each in 4 ⫻ SSC/0.01% Tween solution at 42°C, and then reacted with nitroblue tetrazolium/5-bromocresyl-3-indolylphosphate chromogen (Roche Molecular Biochemicals) for 30 minutes. Sections were rinsed again and counterstained with 0.1% nuclear fast red and analyzed under a light microscope. Some tissue sections were processed without probe to serve as negative controls. SRY⫹ cells were evaluated by counting positive cells per 1000 hepatocytes in each liver.
Statistical Analysis Continuous variables were expressed as mean values ⫾ SD. Differences of continuous parameters among groups were subjected to one-way analysis of variance followed by Scheffe’s post hoc test. A value of P ⬍ .05 was considered significant. Animal survival was evaluated using Kaplan-Meyer plots. The data were analyzed using SPSS software version 11.0 (SPSS Inc, Chicago, Ill, USA).
RESULTS Survival Rate Was Low in PLTx
Graft survivals are shown in Fig 1. In the PLTx group, the rate of survival at 7 days was 5 of 10. In contrast, recipients in the WLTx and PHx groups survived indefinitely. Liver Injury Was Serious in PLTx
On days 7 and 14 postoperatively, livers from the PHx group maintained a normal architecture. Livers from the WLTx group rats demonstrated only scattered hepatocyte necrosis, maintaining a rather normal architecture. In contrast, livers from the PLTx group showed hepatocellular necrosis around the portal tract areas (Fig 2A to 2C).
Fig 3. Serum ALT and AST activity. (A) Serum ALT and (B) AST activities after operation (mean ⫾ SD) (n ⫽ 6). Vertical bars represent the SD from the mean. Statistical significance. #P ⬍ .05 for PLTx versus PHx and WLTx livers at the different time points. *P ⬍ .05 for PLTx versus PHx and WLTx groups at the same time points.
1606
LIU, PAN, CHEN ET AL
Fig 4. Hepatocyte-proliferative activity of liver after operation. Percentage of (A) mitoses and (B) PCNA index (mean ⫾ SD). # P ⬍ .05 for PLTx versus PHx and WLTx at the different time points. *P ⬍ .05 for PLTx liver versus PHx at the same time points.
Serum AST and ALT levels increased on day 1 and decreased on the following days in the PLTx, PHx, and WLTx groups. The PLTx values were significantly higher than those in the PHx or WLTx groups at various times (P ⬍ .05); (Fig 3). These findings suggested that liver injury was more serious in the PLTx group. The Capacity of Liver Regeneration was Lower in PLTx
The percentages of liver regeneration parameters are shown in Fig 4. Compared with the PHx group, the livers in the PLTx group showed significantly lower regeneration rates on day 1 and day 3, and higher regeneration rates on days 7 and 14. The maximum PCNA index of hepatocytes was 65.83% ⫾ 13.00% for PHx rats, 47.00% ⫾ 6.81% for PLTx rats (P ⬍ .05). The cells proliferating at 3 days after PLTx were predominantly in the periportal and midzonal region of the liver (Fig 5A to 5C). These data suggested that regeneration in a partial liver graft was decreased and delayed compared with PHx. HSC Mobilization and Homing to Liver Graft After PLTx
The changes in 2m-/Thy-1.1⫹ and CD34⫹ cells in PB are shown in Fig 6. Compared with WLTx and sham groups, 2m-/Thy-1.1⫹ and CD34⫹ cells in peripheral blood (PB) in PLTx and PHx groups increased on day 1 postoperatively and decreased on the following days. Compared with PLTx group, 2m-/Thy-1.1⫹ and CD34⫹ cells were higher than those in PHx on day 1 (P ⬍ .05). The number of c-kit⫹ and Flt-2/3⫹ cells in PB displayed no change at various times.
Fig 5. Immunohistochemical staining of PCNA-positive nuclei. (A) PLTx group showed lower positive cells number than (B) PHx group. (C) Liver in WLTx showed few positive cells. Brown-stained hepatocytes represent positive staining, indicating hepatocytes in S through M phases of the cell cycle. (Original magnification ⫻200.)
We assessed stem cell migration into liver grafts using sections stained with CD34, c-kit, and Thy-1.1. Staining by all three antibodies on serial sections showed similar patterns: with all staining was located in the periportal region spreading outward, but CD34⫹ cells were also c-kit⫹ and thy1.1⫹ or not. At higher magnification, the reactivity of the antibodies was primarily on infiltrated monocytes, or what other investigators have termed “transitional cells.” Also, in PLTx, a few sinusoidal lining cells and bile ductules were positive for c-kit, whereas in the normal liver, they were negative, as in Fig 7E and Fig 7F with two different staining methods. SRY⫹ Cells in Liver Grafts
In the male rat (positive control), SRY positivity was observed in the majority of hepatocytes (Fig 8A). Contaminating hematopoetic cells, preferentially arranged periportally, were easily distinguished by morphology and excluded from counting. In the PLTx group, few SRY⫹ hepatocytes were observed (Fig 8C). DISCUSSION
It has been reported that bone marrow– derived stem cells can give rise to hepatocytes, oval cells, and bile duct cells.15,17 Therefore, it has been proposed that HSC, can migrate into sites of injury undergoing tissue-specific differentiation that promotes structural and functional repair. To test whether HSC could be mobilized and
HEMATOPOIETIC STEM CELL MOBILIZATION
1607
Fig 6. The change of 2m-/Thy1.1⫹ and CD34⫹ cells in PB in four groups by FACS. The change of (A) 2m-/Thy-1.1⫹ and (B) CD34⫹ cells. *P ⬍ .05 for PLTx and PHx versus WLTx livers at the same time points, respectively.
naturally migrate into the liver after hepatic injury and have the capacity to promote liver repair, we used a sex-mismatched rat PLTx model. In this model donorderived hepatocytes are detected using the Y chromosome as a marker. First, we evaluated the injury and regeneration of the
liver. Compared with PHx and WLTx, the PLTx cases showed worse histology of the liver grafts, lower survival rates were and higher levels of AST and ALT. These results indicated that liver injury in PLTx was more severe. The mitosis index and PCNA in PLTx were lower than those in PHx, which suggested that the capacity of
Fig 7. Immunohistochemical staining of CD34⫹ and c-kit⫹ cells on day 3 after transplantation. The brown-stained CD34⫹ and c-kit⫹ cells were mainly in the portal tract areas. More CD34⫹ cells were found in (A) PLTx than (B) WLTx and more c-kit⫹ cells were found in (C) PLTx than (D) WLTx. The (E) blue-purple and (F) red-stained c-kit⫹ was showed on duct epithelium. (Original magnification ⫻400.)
1608
LIU, PAN, CHEN ET AL
Fig 8. ISH for SRY⫹ cells (bluepurple dot, arrow) in the liver. (A) A male liver specimen showed that there were Y chromosome signals (indicated by arrows) in hepatocytes. (B) A female liver specimen showed that there were no Y chromosome signals in hepatocytes. (C) A femalemale isograft specimen in the PLTx group showed that there were few Y chromosome signals (indicated by arrows) in hepatocytes. (Original magnification [A,C] ⫻400, [B] ⫻300.)
liver graft regeneration was decreased. In PLTx, liver injury was related not only to loss of liver mass but also to reperfusion and early immune responses,21 which may explain the difference between liver injury and regeneration in PLTx versus PHx. Then, we studied the role of HSC mobilization focusing on 2m-/Thy-1.1⫹, CD34⫹, c-kit⫹, and flt-2/3⫹ HSCs, which are precursors of blood and endothelial cells. We observed that CD34⫹ and 2m-/Thy-1.1⫹ cells in PB were increased on day 1 after operation in PLTx and PHx but not in WLTx and sham groups, which indicated that HSC were mobilized, probably because of the loss of liver mass. Howerver, we found CD34⫹ and 2m-/Thy-1.1⫹ cells in PLTx to show higher levels than those in PHx CD34⫹ and Thy-1.1⫹ cells were increased in partial liver grafts, but not in PHx or WLTx. These results indicated that more CD34⫹ and Thy-1.1 cells were mobilized and homed to the liver graft after PLTx, which may depend on a more serious liver graft injury and an increased immune response22 in rat PLTx. In addition, we found c-kit⫹ cells, partly expressed on duct epithelium, increased in the liver graft but not in PB. The c-kit protein (CD117) has been identified as the natural receptor for the cytokine stem cell factor, representing a marker of both HSC and oval cells.23 It has been suggested that activated endogenous liver oval cells may be involved in regeneration. HSC contribution to liver regeneration was evaluated by ISH for SRY in paraffin sections after sex-mismatched transplantation. SRY is a transcriptional activator and although its use as a marker could generate concerns of false negativity in quiescent cells outside the male reproductive system, we provided evidence for SRY detection in resting cells, because the vast majority of cells in male control livers were SRY⫹. We found few SRY⫹ cells in PLTx, which suggested few exogenous cells were involved in regeneration. Analysis of liver regeneration and SRY⫹ cells suggested liver restoration was mainly endogenous, with less support from HSCs, because the majority of proliferating cells were of host, rather than HSC, origin. HSCs, mobilized after PLTx and PHx, may be a normal
phenomenon to respond to stress signals from the injured liver. Studies24 have shown that the chemokine SDF-1 and its receptor CXCR4 participate in the mobilization of HSCs from bone marrow and in the migration of HSCs to the injured liver. However, liver regeneration mainly focused on endogenous hepatic restoration programs. The precise impact of HSC mobilization and migration on partial liver grafts remains unknown.
REFERENCES 1. Kiuchi T, Kasahara M, Uryuhara K, et al: Impact of graft size mismatching on graft prognosis in liver transplantation from living donors. Transplantation 67:321, 1999 2. Lo CM, Fan ST, Liu CL, et al: Minimum graft size for successful living donor liver transplantation. Transplantation 68: 1112, 1999 3. Ferrari G, Cusella-De Angelis G, Coletta M, et al: Muscle regeneration by bone marrow-derived myogenic progenitors. Science 279:1528, 1998 4. Krause DS, Theise ND, Collector MI, et al: Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 105:369, 2001 5. Lin Y, Weisdorf DJ, Solovey A, et al: Origins of circulating endothelial cells and endothelial outgrowth from blood. J Clin Invest 105:71, 2000 6. Asahara T, Murohara T, Sullivan A, et al: Isolation of putative progenitor endothelial cells for angiogenesis. Science 275:964, 1997 7. Brazelton TR, Rossi FM, Keshet Gl, et al: From marrow to brain: expression of neuronal phenotypes in adult mice. Science 290:1775, 2000 8. Mezey E, Chandross KJ, Harta G, et al: Turning blood into brain: cells bearing neuronal antigens generated in vivo from bone marrow. Science 290:1779, 2000 9. Jang YY, Collector MI, Baylin SB, et al: Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 6:532, 2004 10. Harris RG, Herzog EL, Bruscia EM, et al: Lack of a fusion requirement for development of bone marrow-derived epithelia. Science 305:90, 2004 11. Ianus A, Holz GG, Theise ND, et al: In vivo derivation of glucose-competent pancreatic endocrine cells from bone marrow without evidence of cell fusion. J Clin Invest 111:843, 2003 12. Wang X, Willenbring H, Akkari Y, et al: Cell fusion is the principal source of bone marrow-derived hepatocytes. Nature 422:897, 2003
HEMATOPOIETIC STEM CELL MOBILIZATION 13. Vassilopoulos G, Wang PR, Russell DW: Transplanted bone marrow regenerates liver by cell fusion. Nature 422:901, 2003 14. Petersen BE, Bowen WC, Patrene KD, et al: Bone marrow as a potential source of hepatic oval cells. Science 284:1168, 1999 15. Theise ND, Badve S, Saxena R, et al: Derivation of hepatocytes from bone marrow cells of mice after radiation-induced myeloablation. Hepatology 31:235, 2000 16. Theise ND, Nimmakayalu M, Gardner R, et al: Liver from bone marrow in humans. Hepatology 32:11, 2000 17. Korbling M, Katz RL, Khanna A, et al: Hepatocytes and epithelial cells of donor origin in recipients of peripheral-blood stem cells. N Engl J Med 346:738, 2002 18. Alison MR, Poulsom R, Jeffery R, et al: Hepatocytes from non-hepatic adult stem cells. Nature 406:257, 2000 19. Wright DE, Bowman EP, Wagers AJ, et al: Hematopoietic stem cells are uniquely selective in their migratory response to chemokines. J Exp Med 195:1145, 2002
1609 20. Kamada N, Calne RY: A surgical experience with five hundred and thirty liver transplantations in the rat. Surgery 93:64, 1983 21. Dirsch O, Dahmen U, Gu YL, et al: Influence of cold ischemia on liver regeneration after partial liver transplantation. Transplant Proc 34:2303, 2002 22. Omura T, Nakagawa T, Randall HB, et al: Increased immune responses to regenerating partial liver grafts in the rat. J Surg Res 70:34, 1997 23. Matsusaka S, Tsujimura T, Toyosaka A, et al: Role of c-kit receptor tyrosine kinase in development of oval cells in the rat 2-acetylaminofluorene/partial hepatectomy model. Hepatology 29: 670, 1999 24. Kollet O, Shivtiel S, Chen YQ, et al: HGF, SDF-1, and MMP-9 are involved in stress-induced human CD34⫹ stem cell recruitment to the liver. J Clin Invest 112:160, 2003