| Owing to the faster regeneration of both circulating neutrophils and platelets, G-CSF mobilized PBSCs have become the primary source of hematopoietic stem cells for clinical transplantation over the past ten years. Recent studies showed that PBSCs could also transdifferentiate into various tissue cells and be regarded as the potential stem cell resource to regenerate damaged tissues including cirrhotic liver. G-CSF significantly improved survival and liver histology in chemically injured rodents by enhancing the migration of bone marrow (BM)-derived progenitors to liver, as well as promoting endogenous hepatocyte repair mechanisms. Certain clinical studies have reported that both G-CSF treatment and PBSCs transplantation could ameliorate the liver function of patients with end-stage liver disease. We also found previously by clinical surveys that PBSCs transplantation via hepatic artery could improve liver function of patients with HBV-related decompensated liver cirrhosis. However, dut to stem cell diversity in PBSCs, which subgroups contributing to this process remains unexplored.Circulating monocytes are traditionally believed to be committed precursors for phagocytes, such as macrophages and dendritic cells. However, it is recently reported that this cell subgroup contain progenitors with capacity to differentiate into a variety of non-phagocytes, including bone, cartilage, fat, skeletal, cardiac muscle, neuron, endothelium and hepatocyte. We previously reported that human adherent PBSCs predominantly positive for monocyte marker CD14 could differentiate into functional hepatocyte-like cells in vitro or in nude mouse liver. It could be concluded that circulating monocytes are more multipotential than thought previously.Moreover, circulating monocytes have been reported to contribute to liver cirrhosis reversion. Using a transgenic mouse model in which CD11b+ cells could be conditionally depleted, Duffield et al. found that liver-resident monocytes and macrophages were instrumental in the removal of fibrosis during any recovery phase. Fischer et al. also reported that macrophages could also provoke apoptosis of HSCs by the expression of TRAIL. However, little is known about the effect of monocytic progenitors in PBSCs transplantation on liver cirrhosis in vivo.【Objectives】(1) To illustrate the characterization of rat monocytes in PBSCs. (2) To compare the theraputic potential of CD14+ monocytes and CD14- cells in PBSCs for carbon tetrachloride (CCl4)-induced rat liver cirrhosis. (3) To investigated the underlying molecular mechanisms through which CD14+ monocytes participate in injuried liver repair.【Materials and methods】(1) PBSCs of male rat were mobilized by G-CSF and were collected by density centrifugation with Ficoll solution. (2) CD14+ monocytes in PBSCs were separated by magnetic cell sorting (MACS). (3) The characterization of CD14+ monocytes in PBSCs were determined by morphology observation, flow cytometry analysis (FACS) and cytochemistry assay. (4) Female rats with CCl4-induced liver cirrhosis were injected CM-DiI-labeled CD14+ monocytes (1x107 cells/rat) or CD14- cells via the portal vein. (5) Therapeutic potential of transplanted cell for liver cirrhosis was determined by serum and portal venous pressure (PVP) assay. (6) Liver fibrosis was quantified with sirius red staining, masson trichrome staining and immunohistochemical analysis. (7) Distribution of transplanted cells in injuried liver was detected by fluorescence assay and PRINS. (8) The characterization of transplanted cells in injuried liver was detected by confocal microscopy assay. (9) Hepatocyte regeneration was determined by immunohistochemistry and real-time PCR. (10) ECM degradation was determined by in situ fluorescent zymography, gelatin zymographym, western blot, real-time PCR and confocal microscopy assay.【Results】1. Isolation and characterization of rat monocytes in PBSCsRat PBSCs mobilization was induced by G-CSF administration, product of leukapheresis was obtained and CD14+ monocytes were further isolated by MACS. By FACS analysis, the positively isolated cells were predominantly positive of monocyte specific markers, including CD14 (95.4%±0.3%) and CD11b (93.7%±1.8%) . Cytochemistry assay confirmed the expression of CD11b. This subgroup of cells cultured in plastic plates exhibited adherent potential as monocytes. After 3-5 days without other exogenous factors, they manifested clonogenic potential. In addition, monocytes in PBSCs were also positive for markers of hematopoietic stem/progenitor cells, including CD34 (1.1%±0.3%), CD45 (42.8%±4.6%), antigen for mesenchymal stem cells CD44 (50.1%±4.3%), and pluripotency-associated transcription factors Oct3/4 (4.2%±1.1%) and Sox2 (5.7%±1.5%). However, monocytes were negative for CD133, the marker of endothelial stem cells.2. Significance of monocytes in PBSCs for liver cirrhosisThe rats with CCl4-induced liver cirrhosis were divided randomly into 2 treatment groups: (1) monocytes treated; and (2) CD14- cells infused. The rats that underwent both monocyte and CD14- cell transplantation showed a gradually increased ALT and AST level compared with those before transplantation. It should be noted that an elevated serum albumin level and reduced portal vein pressure in monocyte-transplanted rats were only found which contributed to a significantly improved survival rate.2 weeks and 4 weeks after transplantation, the monocyte-transplanted livers apparently showed reduction of liver fibrosis compared with the livers treated with CD14- cells by sirius red staining, masson trichrome staining and immunohistochemical analysis for fibronectin respectively, although small dose of CCl4 administration was maintained throughout the experimental period. Quantitative image analysis indicated that the percentage of liver fibrosis area before monocyte transplantation was 9.3%±1.9%, but 4 weeks after transplantation, the percentage was 2.1%±0.9% in monocyte-transplanted livers compared with 11.2%±2.3% in CD14- cell-infused livers (p < 0.01). In addition, monocyte-transplanted livers had fewerα-SMA-positive and TGF-βproducing cells than did CD14- cell-infused livers. RT-PCR confirmed that monocyte transplantation significantly attenuated the hepatic mRNA expression of fibronectin,α2-(1)-procollagen,α-SMA and TGF-βcompared with those before transplantation or receiving CD14- cells for 1 week (p < 0.01).3. The mechanism of transplanted monocytes promoting hepatocyte regenerationTo trace the transplanted cells, monocytes were stained with red fluorescent marker CM-DiI. 1 week after transplantation, CM-DiI-labeled cells mainly clustered in periportal areas (86.9%±10.1%), whereas seldomly in parenchyma areas (8.4%±2.5%). PRINS showed SRY gene could be detected in female recipient rat livers. Next, we explored which cell lineages could be drived from monocytes. Approximately half of labeled cells (62.1%±9.8%) present in the liver were stained positive for F4/80, a marker of macrophage/Kupffer cells, but negative forα-SMA, a specific marker of myofibroblast. In addition, there was a limited number of labeled cells (1.8%±0.4%) that expressed albumin and CK18, hepatocyte markers. However, none of labeled cells were stained for OV-6, a marker of oval cells.To evaluate whether monocyte transplantation enhance the proliferation of hepatocytes in cirrhotic livers, we assessed the Ki-67 expression levels by immunohistochemistry. In the monocyte-transplanted livers, the percentage of Ki-67-positive cells increased significantly, compared with that in the CD14- cell-infused livers. The percentage increased from 10.79%±2.98% (1 week) to 15.31%±3.24% (2 week) after monocyte transplantation, vs from 6.16%±1.24% to 8.45%±0.98% after CD14- cells injection (p < 0.01). 1 week after monocyte transplantation, livers showed a significant increase of mRNA expression of various growth factors associated with hepatocyte regeneration including HGF, TGF-α, EGF and VEGF, compared with CD14- cell-infused livers (p < 0.01).4. The mechanism of transplanted monocytes reversing liver fibrosisAs described above, we found that established liver fibrosis could be attenuated after monocyte transplantation. Hence, we speculated that MMP might be involved in this regression process. In situ fluorescent zymography demonstrated the fierce elevation of the whole enzymatic activities of MMPs in monocyte-transplanted livers, in contrast to those in CD14- cell-infused livers. We also analyzed the MMP-2 and -9 activities by gelatin zymography. Following monocyte transplantation, activities of both pro- and active MMP-2 and -9 were gradually increasing, much stronger than those in CD14- cell-infused livers. Two weeks after monocyte transplantation, MMP-2 and -9 activity reached peak. In addition, monocyte transplantation promoted not only MMP-2 and -9 protein expression, but also MMP-13 expression, a specific collagenase, in the cirrhotic liver. Monocyte-transplanted livers showed a boost of MMP-2, -9 and -13 mRNA expression, compared with CD14- cell-infused livers at 1 week after transplantation. We also found that the expression of TIMP-1 mRNA decreased following monocyte transplantation by real-time PCR. Finally, we observed transplanted monocytes expressing MMP-9 and -13 by immunofluorescence. It showed that some labeled monocytes could produce MMP-9 and -13, but a lot of indigenous cells in injuried livers could also do that after monocyte transplantation.【Conclusions】In conlusion, CD14+ monocytes in PBSCs contribute to the hepatocyte regeneration and ECM remodeling in rat liver cirrhosis much more than CD14- cells. CD14+ monocytes might be the vital stem cell subgroup of PBSCs for the treatment of end-stage liver disease. |
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