Experimental Study On Long-term Cultivation Of Human Fetal Hepatic Stem Cell In Vitro

Background At present the incidence rate of acute or chronic liver failure is still very high in our country,its pathological feature is mass necrosis of hepatocytes, liver regeneration is inhibited by many factors.OLT is an available effective therapy for terminal liver failure. But the extensive application is restricted due to some factors, such as increasing shortage of donor livers, high cost, using immunosuppressant all life, and so on.Transplantation of hepatocytes is an important supplement for OLT as auxiliary therapy.There are some problems unsolved ,such as the source and number of hepatocytes, its proliferative potential, functional characteristics. Normal human hepatocytes are ideal source for cell transplantation theoretically,but its application is also limited by organ availability and difficulty in proliferation; Heterogeneous hepatocytes are of extensive sources, with immunologic rejection and virus infection, especially retrovirus infection; Neoplastic cell strain of liver can proliferate powerfully, taking the risk of neoplasia, and the function of hepatocytes is weak; Although immortalized cell line (mostly by transferring SV40 LT ) can solve proliferative problem, its safety is doubted.Growth factor secreted by human fetal hepatocytes can stimulate cellproliferation in vitro. Human fetal hepatocytes are suitable sourceswith weak immunogenicity, strong differential and proliferative potential; Clinical data indicates human fetal hepatocytes may be useful for treating hepatic failure and genetic enzymatic deficiency,whereas exist above-mentioned problem. Hepatic stem cells can slove these problems. HSC are considered to be a kind of bipotential cells with proliferative capability, cell number of transplantation is only 1/3 to 1/5 of mature hepatocytes. Fetal hepatic stem cells are small in volume, ease to transplant through portal vein, integrate into hepatic plate and gene operation.It has been estimated that stem cells in quiescet adult liver are limitary, while there are plenty of stem cells in embryonic liver. If we choose human fetal hepatic stem cell as the source of cell transplantation, we will avoid the infection of unknown pathogens and spread of heterogen in the crowd. Thus, the isolation purification and proliferation of hepatic stem cells successfully in vitro are of significance.Our experiment made elementary investigation on the separation and proliferation of hepatic stem cell, aiming to explore the method of long-term cultivation. This experiment is devided into two parts. The first part is about separation, purification, cultivation and identification of hepatic stem cells, to obtain stem cells with high viability and more purity. In the second part we use different combinations of growth factors to accelerate proliferation of hepatic stem cells.We want to prove the possibility and effectivity of this way, and possibile mechanisms. The experiment will pave the way for further study and clinical application.Part I Isolatioru Cultivation and Identification of HumanFetal Hepatic Stem CellObjective To study the isolation, purification and identification of human fetal hepatic stem cells. Methods We developed a procedure utilizing enzymatic digestion (collagenase type IV) and Percoll discontinuous desity gradient centrif iguation to isolate human fetal hepatic stem cell. Livers come from aborted fetus of 4 to 6 months gestation, liver cell suspensions were obtained by umbilical vein perfusion digestion, then used Percoll gradient (1.110,1.070,1.035g/ml) to centrifugate the liver cells suspension. Extracted the cells (HSCs) between 1. 110 and 1. 070g/ml Percoll, and washed twice with PBS. Cell yield was determined using counting chamber. Cell viability was determined using trypanblue exclusion assay. Then adjusted the density of cells(lX105) and cultured them in DMEM/F12 media. The morphologic character of human hepatic stem cells was observed by optic and electronic microscopy. The surface markers of HSCs such as AFP, ALB, CK19> CK8, CD34, VIMETIN were identified by ICC. Counted the amount of the cells of different branches during the whole process. Computed the yield of human HSCs and estimated the enrichment of human HSCs in human fetal liver. The data was dealt with by SPSS 11.5 for Windows. Results The connective tissue of fetal liver had been digested effectively using two-step collagenase purfusion, and liver cells could be separated easily. After the liver cells suspension was centrifugated in discontinuous dentity Percoll solution, the HSCs could be extracted and purified from liver cells. The mean viability of HSCs was (90.80±2. 04)% by trypanblue exclusion assay many times. With optic.microscopy we canobserve that HSCs had oval appearance and were less than 1/3 to 1/5 size of normal hepatocytes. With electronic microscopy we found that HSC was approximately 7—13fim in diameter, there were some protruding microvilli on surface, the oval nucleus occupies the most space of the cell and there were few organelles, a high nuclear to cytoplasm ratio, endoplasmic reticulunu mitochondrias ribosomes and other organelle, indicating young undifferentiated cellular characteristic. Immunocytochemical staining showedrthe freshly isolated cells coexpressed AFP, CK19> CK8^ CD34, ALB and VIMETIN , not expressing LCA. By counting the amount of cells and weighing the livers,, the mean yield of HSCs was (2. 74 + 0. 77) X107 per liver(n=10).The enrichment of HSCs was about (5.98±0.95) X105/g(n=10). In primary culture addherent time was 2 days to 3 days, we can observe small fusiform cells,or more angular cells, the medium was changed every 3 days, loosely adherent cells were removed. HSC proliferate with the generation of more cells, gradually migrated and interacted of cells, to form cell clusters,like epithelial cells.HSCs were subcultured for the second passage on day 6 or 7 post isolation. Conclusions Satisfactory HSCs can be obtained by enzymatic digestion and centrifugation of discontinuous dentity Percoll solution. Obtained HSCs have same morphologic and ultrastructural characteristics which have been mentioned in references. Immunocytochemical staining shows that HSCs are positive for surface markers AFP> CK19, CK8, CD34> ALB, VIMETIN, but negative for LCA which is the marker of lymphocyte. The quantity and viability of HSCs is considerable, and HSC can proliferate in vitro, displaying the potential for serial passaging. We can use them for the later experiment.Key words human fetal hepatic stem cell; cell isolation; cell culture;cell identificationPart II Effects of Growth Factor on Proliferation of HumanFetal Hepatic Stem CellObjective To evalute the effects of different growth factor combinations on proliferation of HSC and to establish culture system of human fetal hepatic stem cell. Methods HSCs were obtained by using Percoll gradient centrifugation, and then selected by adherence method. HSCs were placed in 24-hole tissue plates(3X10Vml),and cultured under different conditions (group I ^ IK IIK IV). Group I was used as the control (without growth factor), rhLIF was used in group II,rhHGF and rhLIF were used in group III, rhHGF^ rhSCF and rhLIF were used in group IV.MTT was used to detected the level of growth factors on proliferation of HSCs cultured for primary 2^ primary 3^ primary 5. The variation of cell cycle was detected with the flow cytometry(FCM) with the third generation cells,and morphological changes were observed with microscope and microphoto -graphs. At the same time, The makers of HSCs such as AFP% ALB^ CK19> CK8n CD34 were identified by ICC, counting positive cells, computing positive rate. Data was processed and analysed by SPSS for Windows 11.5, then the best combination was used to culture HSCs. Result Under the same celluar detensity and cultivate time, cell proliferated slowly in control group, there was no obvious effect on proliferation of HSC in group II (P>0. 05), but subcultured more generations; cell can proliferate in group III obviously (P<0. 05), the percentage of cells from phase G0+G1 to phase S increased; group IV can also accelerate the proliferation of HSCs (p<0.01), the number of clone formation increased, the percentage of cells from phase G0+G1 to phase S increased. The combinations of rhSCF^ rhHGF^ rhLIF were also...