Applying Green Fluorescence Protein To Trace The Fetal Liver Stem/progenitor Cells Enriched By Percoll Centrifugations

Since the donor for liver transplantation is limited, more and more attention is focusing on the possibility of cell transplantation. The ultimate goal for successful cell transplantation is to find cell populations that can effectively proliferate and replace functional liver mass without genetic manipulation or major damage to the recipient liver. So far, fetal liver cells are one of the cell sources identified that fulfill all the criteria of above mentioned, which can effectively repopulate the normal liver. However, the complicated components of fetal liver and the unknown surface markers of liver stem cells (LSCs), hinder the isolation and application of FLSPCs. This research combined Percoll discontinuous gradient centrifugation (PDGC) with Percoll continuous gradient centrifugation (PCGC) to enrich rat fetal liver stem/progenitor cells (FLSPCs). After PDGC selection, purification of FLSPCs in culture by differential trypsinization was employed. By flow cytometry analyses (CD117, CD49f, c-Met, Sca-1, CD90, CD105, and CD133), it revealed the existence of LSCs and a heterogeneous cell population. For first-time PDGC enrichment, FLSPCs-like cells might be up to 50%; for second-time PCGC enrichment, the purification might get to 73%. Besides, we propose that combining several markers, such as CD117, CD49f, Sca-1 and c-Met, may obtain genuine stem cells. To trace the exact effect of FLSPCs on amending liver injury, by liposome-mediated transfection, the green fluorescent protein (GFP) gene was transferred into the FLSPCs. After the FLSPCs were successfully transfected by pAcGFP1-N1 plasmid, they were transplanted into the same strain rats through portal vein injection. Finally, the effect of transplanted FLSPCs on liver regeneration and improving acute liver injury was observed. To conclude, the combination of PDGC with PCGC techniques may contribute to promote the important biological aspect of LSCs isolation, the FLSPCs with GFP labeling will provide an useful source for cell transplantation research.Aims :1. The enrichment and identification of FLSPCs: apply PDGC and PCGC to enrich FLSPCs, and use flow cytometry to identify FLSPCs.2. The amplication and purification of FLSPCs: use differential trypsinization and attaching to purify FLSPCs, then construct the proliferating system for FLSPCs3. The tracement of FLSPCs: through liposome-mediated transfection, the FLSPCs were stedily labeled by GFP.4. The amending effect of FLSPCs: transplant FLSPCs into acute liver injured rats, and observe the effect of FLSPCs on improving the liver injury. Methods:1. The enrichment and identification of FLSPCsThe fetal liver of ED 14 d rat was trypsinized for isolation of fetal liver cells. The obtained cells were subject to PDGC for first-time enrichment of FLSPCs, and the cell layer between the 50% and 70% Percoll solutions was selected. By flow cytometry (FCM) analyses (CD117, CD49f, c-Met, Sca-1, CD90, CD105, and CD133), the existence of FLSPCs was examined. To advancedly enrich the FLSPCs, the PCGC was used to fractionate the first-time enriched cells. Similarly, the profile of FLSPCs was valued by FCM.2. The amplication and purification of FLSPCsWhen cells being cultured, the FLSPCs were purified by a two-step method. As the cells were firstly trypsinized with 0.25% trypsin containing EDTA in 5 min, the floating cells were droped; then the remaining cells were totally trypsinized for subculture, the big cells which couldn't attach the plate in 100 min were droped. After the two-step purification, the remaining cells were cultured in complete Williams'E culture media supplied with stem cell growth factor.3. The tracement of FLSPCsThe plasmid DNA of pAcGFP1-N1 was extracted from TOP10 E coli and identified by electrophoresis. By liposome-mediated transfection, the GFP gene was transferred into FLSPCs. Using neomycin screening, the steadily transfected FLSPCs were selected.4. The amending effect of FLSPCsThirty SD rats of the same line were randomly divided into three groups: blank control group (A), CCl4 injured group (B) and FLSPCs transplantation group (C). Rats in group A were intraperitoneally injected with normal saline, while rats in group B and C were intraperitoneally injected with CCl4. Twenty-four hours later, all rats were anaesthetized by intraperitoneally injection of butaylone, then group A and B were injected with normal saline via portal vein, and group C were injected with FLSPCs suspension. 4 weeks later, the levels of serous ALT and AST in each group were tested, and pathological situations of livers were evaluated.Results:1. By FCM analyses, it reveals the expression profiles of stem cell markers in first-time enriched FLSPCs by PDGC were all above 50%, including the most widely used markers CD133 and CD49f. Especially, more than 85% cells expressed CD117 and C-Met. That is to say that the enriched cells were heterogeneous, which was consistent with the results of morphology. After the cells were successfully layered by PCGC, the properties of stem cells were valued in each fraction. As the number of fraction grew bigger and bigger, the sizes of cells became bigger and bigger, the expressions of CD133 and CD49f decreased gradually(P<0.05). What's important, in the forth layer of cells there was a sharp reduction of markers expressions, which indicated the existence of mature cell populations. Using two-step enrichment, more than 73% FLSPCs expressed both CD133 and CD49f.2. As the enriched FLSPCs were cultured in plates, the sizes of cells displayed heterogeneous. Being trypsinized in 5 min, the small stem-like cells remain former shapes, however, big cells became round, detached from plates, and were droped. When the cells were being subcltured, the small cells could attach the plates in 100 min, in contrast, the big cells couldn't. Thus the big cells were droped. After the two-step purification, the FLSPCs became highly homogeneous. 3. Four hours after the FLSPCs were transfected pAcGFP1-N1 plasmid, several cells displayed green under fluorescence microscope. Ten days later, about 20% cells displayed green., and the cells uniformly distributed. Another 10 days later, 40% cultured cells gave out green fluorescence. About one month later, the transfection efficiency was evaluated as 60%. As the selection pressure of neomycin were applied, the untransfected cells were gradually died and replaced by transfected cells.4.Serous ALT and AST of group A were (12.58±6.32) U/L and (13.18±5.73) U/L, while those of group B were (190.23±13.13) U/L and (172.34±11.09) U/L, which were obviously higher than those of group A (P<0.01). Serous ALT and AST of group C were (110.52±9.73) U/L and (106.02±10.36) U/L, which were higher than those of group A (P<0.01) and lower than those of group B (P<0.01). The significant differences of ALT and AST levels in A, B and C groups indicated two important aspects: one side the injury model was successful; the other side, transplanting FLSPCs could effectively improve the situation of acute liver injury caused by CCl4. The degrees of pathological injury were significantly different either. Pathological section of group B displayed a lot of inflammatory cells, large death areas of hepatic cells and part of hepatic plates are injured. Whereas, pathological section of group C displayed fewer inflammatory cells and edema hepatic cells, slighter fatty degeneration of hepatic cells and the hepatic plates were complete.Conclusion:1.A systematic method for effectively enriching and purifying FLSPCs is successfully constructed. The method can not only provide the enough quatity of FLSPCs, but also adjust the purification of FLSPCs.2. The FLSPCs labeled with GFP are constructed, then it is possible to trace the effects of FLSPCs on liver regeneration in vivo and differentiation in vitro.3. Transplantation of FLSPCs through portal vein has therapeutic effect on acute liver injury induced by CCl4.