The Studies On Isolation And Cultivation And The Mechanisms Of Proliferation And Differentiation Of Human Fetal Hepatic Stem Cells In Vitro

Objective The stem cell studies are thought to gain a breakthrough in the fields of biology and medicine in the new century , which makes human beings have hopes to conquer the diseases. The effective treatments for the patients suffering final stage liver diseases including severe hepatitis, cirrhosis and hepatoma are still very difficult. At present, orthotopic liver transplantation(OLT) should be the most effective method to treat final stage liver diseases, but it is restricted due to the expensive application, clinical shortage of donor organs and immunological rejection and so on. In recent years, hepatic stem cell studies have been further developed, and it has been as a good resource for hepatocyte transplantation. Hepatic stem cells have the strong reproductive activity, and they can differentiate into hepatocytes and bile duct cells. The human hepatic stem cells are the best choice for the clinical treatment, but the most current studies on hepatic stem cells have carried on the animals, so it has important significance to obtain hepatic stem cells from human fetus. These cells should not only have a powerful ability on the differentiation toward mature hepatocytes, but also be suitable to be transplanted and be as the carrier of gene treatment. Hepatic stem cells transplantation is a good measure for the treatment on liver diseases on the terminal stage and it is also an alternative treatment scheme just like OLT.Our study is aim at separating and purifying hepatic stem cells from human fetus liver, and identify its biological functions. Meanwhile, we will detect their abilities of differentiation into mature hepatocytes and the mechanisms of proliferation and differentiation. Our work will supply theory basis for the future cells transplantation and human liver tissues reconstruction in mice and even large animals.Methods and results1. The studies on isolation and cultivation and analysis on the biological characteristics of human fetal hepatic stem cells We isolated stem cells from aborted human fetus by drugs in the early stage of gestation in the methods of associated enzymatic digestion, gravity sedimentation, monoclonal digestion, selective digestion. The sepatation method is simple and operated easily to keep the cells high vigor, which will create good foundations for the cells transplantation and as the carrier of gene treatment. The hFHSCs have the following characteristics:(1) hFHSCs had homogenous small fusiform shape. The hFHSCs were small in size with round nuclear and 1-3 nucleoli. Ultrastructures were observed high cytoplasm to nuclear ratio and few organelles in the cytoplasm by electron microscope.(2) hFHSCs of each generation were in the growth latent period on the first 1-2 days, and their logarithm multiplication period lasted about 3 days. The cell line had been stably propagated in culture up to 10 passages. They were passaged one time every 4-5 days. The doubing time was about 29.32h. The cells had high vigor.(3) It had been demonstrated that they had a normal karyotype, and no tumors formed three months after they were injected subcutaneously into SCID mice. (4) The hFHSCs expressed various kinds of markers. Immunocytochemistry was used to detect their relative protein expression profile. The results showed that they expressed liver-connectors markers such as AFP and ALB, biliary tree markers such as CK19, the common markers on the liver-connectors and biliary tree such as CK-8 and c-met, ovule cells markers such as Thy-1 and c-kit. Using RT-PCR, we detected that hFHSCs expressed AFP,ALB,CK19,CK-8,c-met,c-kit,Thy-1 and didn't express vascular endothelial cell markers such as VE-cadherin, stellate cell marker desmin, hematopoietic cell markers such as CD45 and fetal liver interstitial cells markers such as flk-1.2. The studies on the influence on the proliferation and differentiation into hepatocytes from cell factors in hFHSCsHGF and EGF play key roles in the liver development and regeneration and activation, proliferation, differentiation, migration of hepatic stem cells. In this part, we observed the proliferation and differentiation of hFHSCs resulting from them, which will contribute to building a foundation of identifying the cultivation condition for proliferation and differentiation into mature hepatocytes. Furhtermore, cells and regulatory factors are the two essential elements in the field of liver tissue engineering, and our study will supply theory basis for reconstruction of human liver tissues. Meanwhile, if we can induce hFHSCs to differentiate into mature hepatocytes successfully in vitro, it will be further proved that hFHSCs are the initial cells of liver.(1) We detected that it had the expression of c-met, EGFR, EGF and HGF had not been noted in the hFHSCs by Western blot.(2) Detection of the influence on the proliferation of hFHSCs resulting from HGF: we divided hFHSCs on the same condition into five groups—negtive group: elementary culture solution, HGF(5ng/mL)group: elementary culture solution+ HGF(5ng/mL), HGF(10ng/mL)group: elementary culture solution+ HGF(10ng/mL), HGF(20ng/mL)group: elementary culture solution+ HGF(20ng/mL), HGF(40ng/mL)group: elementary culture solution+ HGF(40ng/mL). By MTT colorimetry, we observed dose-effect relation of HGF in five groups.The results demonstrated that the cells stimulated with higher dose of HGF have higher OD numerical value than negative control group. In the same group, with the time went by, the difference was more significant campared with the value of the 0th day.(3) Detection of the influence on the proliferation of hFHSCs resulting from EGF: we divided hFHSCs on the same condition into five groups—negtive group: elementary culture solution, EGF(5ng/mL)group: elementary culture solution+ EGF(5ng/mL), EGF(10ng/mL)group: elementary culture solution+ EGF(10ng/mL), EGF(20ng/mL)group: elementary culture solution+ EGF(20ng/mL), EGF(40ng/mL)group: elementary culture solution+ EGF(40ng/mL). By MTT colorimetry, we observed dose-effect relation of EGF in five groups.The results demonstrated that the cells stimulated with higher dose of EGF have higher OD numerical value than negative control group. In the same group, with the time went by, the difference was more significant campared with the value of the 0th day.(4) HGF and EGF in conditional culture media induced hFHSCs to differentiate into mature hepatocytes: In this experiment, we divided hFHSCs on the same condition into four groups-negative control group: elementary culture solution, HGF group: elementary culture solution+ HGF(40ng/mL), EGF group: elementary culture solution+ EGF(40ng/mL)and HGF+EGF group: elementary culture solution+ HGF(40ng/mL)+ EGF(40ng/Ml). Every group cells gradually adhered and their shape and size were in uniform. With the time went by, cells number were increasing with cells division growth. However, cells shape in the control and EGF group obviously changed besides the increasing of cells number. In the HGF+EGF group, cells changed not only on the increased number but also on the shape. After hFHSCs were cultivated a week, cells shape begun to change which were demonstrated that they were lenient, abundant kytoplasm. On the 9th day, hFHSCs took on polygon and had more plentiful kytoplasm. From the 12th to 15th day, hFHSCs differentiated into mature hepatocytes with lenient polygon and 2 or 3 nuclears. The changes in the HGF group were similar to the ones in the HGF+EGF group. Every three days replaced the media and collected the specimens to be measured. The content of urea in supernatant of every sample by colorimetry was measured. The results of measured urea showed that the content of urea in supernatant of HGF group and HGF+EGF group increased gradually, especially for the later, while the content of urea in supernatant of control group and EGF group had no obvious difference. We detected the expression of mRNA of ALB and AFP from defferent samples by RT-PCR. By measuring the enrichment of AFPmRNA or ALBmRNA, we found the expression of AFPmRNA decreased and ALBmRNA increased gradually in the HGF+EGF group. The changes in the HGF group was similar to the one in the HGF+EGF group. Whether the expression of AFPmRNA or ALBmRNA, their changes can not be noted not only in the EGF group but also in control group.3. The study on the mechanisms for HGF-mediated proliferation and differentiation in hFHSCsHGF binding to c-met will lead to receptor conformational changes which result in phosphorylation of signal transducers inside different cells. The final result is make cells split, proliferate, differentiate and separate and so on. In our study, we showed that HGF could cause activation of NF-κB in hFHSCs, which may be oneof mechanisms for HGF-mediated proliferation and differentiation in hFHSCs. (1) Using ELASA, we found that it had a positive relation betweent NF-κB activity and HGF dose, With the HGF dose increasing, NF-κB displayed higher activity.(2) By immunocytochemistry staining, nuclear staining was demonstrated, and it had a positive relation with EGF dose. The results Validated intuitively the ELASA results.(3) By RT-PCR, we detected the expresson of P65, P50 and IκBmRNA induced by 0ng/mL or 40ng/mLHGF. The results revealed that in the 40ng/mL HGF group, the expression of P65 and P50mRNA was increased, but the expression of IκBmRNA was weak compared to in the 0ng/mL HGF group, which promoted NF-κB nuclear shifting.(4) Using EMSA, we examined the changes of NF-κB activity simulated by HGF(40ng/mL) in hFHSCs. The results showed that NF-κB activity begun to increase from HGF-treated hFHSCs in 15min and achieved peak time in 60min. Whereafter, NF-κB activity gradually tapered. To confirm specificity of lag band, competition trial was preformed. We found that obvious lag band was formed by binding between marked probes and nuclear extracts and excessive marked NF-κB probes checked this affect, but excessive marked Oct2A probes had no such effect. It was proved that the protein binding to DNA was NF-κB.(5) Using western blot, we examined phosphorylation and degradation of IκBα, a critical regulator which controls nuclear translocation of NF-κB, in the hFHSCs simulated by HGF at diffenert time points-0min, 5min, 10min, 20min, 30min, 60min. The results demonstrated HGF indeed promoted the phosphorylation of IκBαprotein. In 5min after HGF stimulation, phosphorylation of IκBαhad begun. Phosphorylation of IκBαreached the maximum of level in 15-20min after HGF stimulation. After this, the degree of phosphorylation of IκBαbecame weak. Degradation of IκBαwas detedted within 10min and the maximal IκBαdegradation was determinied in 20min after HGF stimulation. These results suggest that HGF binding to c-met on the membrane of hFHSCs would make some relative kinase activate, which promotes phosphorylation of IκBα. And then, NF-κB occurs nuclear shifting and binds to corresponding target gene.Conclusions (1) By the combination of united enzymatic digestion, gravity sedimentation, monoclonal digestion, selective digestion, we isolated hepatic stem cells from human fetal liver in the early stage of gestation. The isolation method is simple and operated easily, and obtained cells have high vigor. (2) hFHSCs accord with foundamental characteristics of hepatic stem cells: they have strong proliferation ability in vitro, express both liver-connectors and biliary tree markers and have the normal karyotype. (3) EGF,EGF-R and c-met proteins are expressed in hFHSCs, and HGF is not noted, which indicats that HGF binding to c-met is by paracrine secretion pathway. (4) HGF and EGF can stimulate hFHSCs to proliferate obviously. (6) HGF plays an important role on inducing hFHSCs to differeniate into mature hepatocytes, and EGF can be in coordination with it. (6) Inducing differentiation system was constructed in vitro, which will contribute to solving the problem of donator shortage on human mature hepatocytes treatment. (7) NF-κB activity was repuired for HGF-induced proliferation and differentiation in hFHSCs by phosphorylation and degradation of IκBα.In summary, we obtained hFHSCs by a simple method and constructed an effective inducing–differeniating cultivation system, meanwhile, the investigations on the mechanisms of proliferation and differentiation in hFHSCs will provide the rationale for optimizing this system. The whole work will provide experiment basis and new clues for solving the problem of donator shortage of human mature hepatocytes and making hepatocytes and hepatic stem cells transplatation come into realities on the stage of clinical treatment.