Experimental Study Of Isolation, Culture And Hepatic Differentiation Of Mouse Embryonic Germ Cell

In 1981 Evans established mouse embryonic stem cell line. Thomson and Gearhart established human embryonic stem cell line from inner cell mass (ICM) of blastocyst and primordial germ cell (PGC) from the fetal gonadol ridges in 1998. Since embryonic stem cells (ES) possess the characteristics of self-renewal and the ability to differentiate into one or more specialized cell types which attract the widely attention in the world. Embryonic stem cell becomes the research hotspot in broad field. The emphases of research is about the isolation , culture and direct differentiation of embryonic stem cells. PGC also is termed embryonic germ cell (EGC),.EGC is much more and easier to obtained than the ES from ICM. So we research systematically on the isolation , culture and direct differentiation to hepatocytes . We have 5 theses in press.一, Isolation, culture and expanding of embryonic germ cell: Isolation, culture and expanding of EGC is the premise of the relative experiment, so we carried out this part firstly ( reference to chapter 1 of the dissertation ).ES are apt to differentiate in vitro and lose their multipotental. In order to maintain the EGC in undifferentiated condition and proliferate raidly, the optional culture system is the key step for the culturing and expanding of EGC in vitro. Weanalyzed the effects of 5 different culture system on the growth , proliferation and its clone form of EGC. The results showed that without feeder layers EGC could not proliferate rapidly in the fibroblast conditioned medium and in the DMEM/F12 medium with or without LIF and it is difficult to passage . EGC could grow and proliferate well on the embryonic fibroblast feeder layers or co-cultured with the mouse embryonic fibroblast. To attach the feeder cells is the premise for EGC to growth and proliferate . So we believed co-culture with embryonic fibroblast is the easr and effective system for EGC isolation , culture and expanding in vitro. We found the best proliferating period of EGC was in the 3rd passage ( reference to section 1 of chapter 1 ).We also cultured the gonadol ridges tissue in DMEM/F12 medium without LIF. The EGC could outgrowth and proliferate rapidly from the gonadol tissue, could form large clone in sheet on the feeder cells. We could obtain much more EGC in this way, but EGC is easy to differentiate in such a culture system (reference to section 2 of chapter 1).— N The research on the directional differentiation of embryonic germ cell into hepatocytes in vitro (reference to chapter 2 of the dissertation)To obtain hepatocytes from embryonic germ cell, it is essential to establish not only an appropriate culture system to induce embryonic germ cell to differentiate into hepatocytes, but also a useful method for identifying differentiated hepatocytes. One of major functions of the liver is elimination of various endogenous and exogenous compounds from the circulation. This clearance process involves basolateral membrane transport systems that mediate the hepatocellular uptake of organic anion and organic caions. Indocyanine green (ICG) is an organic anion that is clinically used as a test substance to evaluate liver function since it is nontoxic and eliminated exclusively by hepatocytes .In the present study , we examined the cellular uptake of ICG to identify differentiated hepatocytes from embryonic liver tissue and differentiated cells derived from embryonic germ cell induced by co-cultured with mouse embryonic liver tissue. The result showed that the ICG were intaken by the differentiated cells which were star and oval in shape, especially ovalcells o The ALB and AAT were also expressed in those differentiated cells. ICG-positive cells possess characteristics of hepatocytes, ICG intake test is an useful way to identify hepatic cells (reference to section 1 of chapter 2 ).To investigate whether EGC can give rise to hepatocytes and to explore the optimal condition under which ES can be induced to differentiate into hepatocytes in vitro, EGC were cultured in the medium with some growth factors ( such as EGF, bFGF, 3 -NGF, RA), liver hepatocyte abstraction and were co-cultured with embryonic hepatocytes. The results showed that by the addition of P-NGF and hepatocyte abstraction and co-cultured with mouse embryonic hepatic tissue , EGC could be effectively induced to differentiate into hepatic cells which could intake ICG, especially oval cells . The ALB and AAT positive expression were also detected in those ICG-positive differentiated cells.This indicated that P-NGF and some factors from hepatocytes could effectively induce EGC to differentiate into hepatocyte-like cells (reference to section 2 of chapter 2 ).Many growth factors in culture medium are closely associated with proliferation and maintaince of an undifferentiated state of ES . Fetal calf serum is usually added to media for ES culture and is used as a source of growth factors. However, some factors in FCS also promote differentiation of ES, which may make it difficult to analyze which one is the true inducer. so we put forward to an inductive protocol without serum to realize the directional differentiation of EGC into hepatocytes .We culture the EGC in B27 serum-free medium added the cytokines such as EGF, p-NGF, RA and hepatocyte abstraction respectively. We found that after the addition of P-NGF and hepatocyte abstraction, The differentiated cells which like star and oval derived from EGC could intake ICG and express the ALB and AAT. But EGF and RA had no such effects. However RA could improve the ratio of differentiated hepatic cells induced by p-NGF and hepatocyte abstraction. This indicated that P-NGF and some factors from hepatocytes could effectively induce EGC to differentiate into hepatic cells in serum-free culture (reference to section 3 of chapter 2).zr.N The research on the differentiation of embryonic germ cell into hepatocytes in vivo (reference to chapter 3 of the dissertation). It is assumed that the microenviroment in vivo play a significant role on the differentiation of these cells.,in which the specific molecules, growth factors, cytokines , interactions between the stem cells and host cells and extracellular matrix are presented to the stem cells in a temporal and sequential manner in order to induce the expression of related developmental genes in sequence and driving their differentiation into different cells. On the basis this theory, we transplanted EGC into normal mice liver and damaged liver by CCL4, then observed differentiation of embryonic germ cell (reference to chapter 3 of the dissertation ).We first implanted embryonic germ cells labelled with BrdU into normal liver by tail vein to test whether embryonic germ cell could undergo milieu-dependent differentiation and express hepatocyte phenotypes in vivo. Two and four weeks after implantation, the transplanted cells were found to be incorporated into the host liver and expressed hepatic-specific proteins albumin (ALB) and synthesed glycogen. We suggested that the normal hepatic microenvironment may induce embryonic germ cells differentiation into hepatocytes. (reference to section 1 of chapter 3).The use of ES in regenerative medicine is attractive as a potantial approach to curing patients with severe disease. To evaluated the engraftment, hepatic differentiation of EGC, and their survival in damaged liver, we established acute and chronic liver-damaged model by CCL4, then allotransplanted EGC into liver-damaged mice. Two and four weeks after implantation, the transplanted cells were found to be incorporated into the host liver and expressed hepatic-specific proteins ALB and synthesed glycogen., the strucure of liver were recovered .We suggested that the damaged hepatic microenvironment may also induce EGC differentiation into hepatocytes and facilitaty the recover from damaged condition ( reference to section 1 of chapter 3).