The Proliferation And Differentiation Of Hepatic Stem Cells And The Regulation Factors

Owing to their multilineage differentiation potential and self-renewing capability, hepatic stem cells might be a source of cells for transplantation of hepatocytes or production of bio-artificial liver. So, hepatic stem cell research is currently "hot" topic.Dabeva reported that hepatic stem cells existed as oval cells in adult liver, which was little in number and difficult to isolate and purify. And it has not been identified about the derivation and the position of oval cells. Alison and Fausto observed that oval cells were activated under conditions such as only injury, carcinoma, and partial hepatectomy So, Alison suspected the existence of hepatic stem cells, supposed that they were hepatocytes which were in special state.It is known that all organs originate from stem cells existing in the anlage of the organ, so hepatic stem cells play a vital role in the formation of liver. Formation and change of liver at the different stages would present the process of proliferation and differentiation of hepatic stem cells. The development of hepatic stem cells is concurrent with the development of the hematopoietic cells and the formation of liver blood vessels. Meanwhile, the development of liver is associated with septum transversum mesenchyme and primitive heart, so, the niche of hepatic stem cells development consist of hematopoietic cells, endothelium, septum transversum mesenchyme and primitive heart. Researchers could study the relationship between hepatic stem cells and those organs through observingchanges of the growth factors and their receptors secreted by those organs. Contrast with investigation in vitro, which only reflects the effect of a factor, studying series sections of embryos would obtain the information of influences of other organs and many factors on hepatic stem cellsHepatic stem cells had been isolated from the liver of mouse, rat, macaque and human. However there are diversities about the time of hepatic stem cells differentiation. Germain reported that hepatic stem cells differentiate to hepatocyte and biliary epithelial cells at the 15d of mouse embryo, while Medlock found hepatic stem cells began to differentiate at the 16-17d of mouse embryo. Meanwhile, Hauruna revealed hepatic stem cells differentiate to hepatocyte and biliary epithelial cells at the 14-16W of human embryo.It has been reported that the signals from heart could induce the expression of liver specific genes (alb and afp), and FGF1, 2, 8 plays an important role to initiate the specific genes expression of hepatic stem cells. Endotheliums interact with newly specified hepatic stem cells, prior to liver bud emergence, and promote hepatic morphogenesis. Expression of some liver specific genes was demonstrated be affected by hepatic transcriptional factors. Using mice models of gene Knock-out, Parviz and others had found that some hepatic transcriptional factors, such as HNF4a, HNF6, HEX and C/EBPa, affect the proliferation and differentiation of hepatic stem cells. However, there is less report about the characteristics, distribution and differentiation of hepatic stem cells. Meanwhile, there is no much informayion about the factors by which the primitive heart, septum transversum mesenchyme, and endothelium might induce liver development. And it has not been reported that the effect of hematopoietic cells on the development of hepatic stem cells. Especially, there is few data about the human hepatic stem cells, which could direct the application research of the hepatic stem cells. To comprehend the biology characteristic of hepatic stem cells and factors that affect the proliferationand differentiation of hepatic stem cell, we are going to study the followingcontents on human embryo and fetus:? The temporal and spatial distribution, morphology, the phenotypes, anddifferentiation of hepatic stem cell. ? The temporal and spatial distribution of factors secreted by the cardiaccells, septum transversum mesenchyme, endothelium and hematopoieticcells, which might regulate the proliferation and differentiation ofhepatic stem cell. (3) The temporal and spatial distribution of factors produced by hepaticstem cells themselves that might affect the proliferation anddifferentiation of hepatic stem cells ? The expression and distribution of HNF4a and HNF6 during theproliferation and differentiation of hepatic stem cell.1. Methods and materials1.1 Collecting of samples and judging the age of gestationA series of 5 urn sections were made from 228 specimens of embryos and futes ranging from 3 ~ 12W. The hematoxylin-eosin staining was performed on one section every ten. The age of gestation was determined under light-microscopy examination according to the number of somites and the state of organs development.2 Morphologic observations? Observing the relationship of embryonic liver and septum transversum mesenchyme and primitive heart.(2) Observing the features of morphology, proliferation and differentiationof hepatic stem cell(3) Observing the morphological change, proliferation and differentiation ofendothelium, hematopoietic cells, cardiac cells and septum transversum mesenchyme.3. Immunohistochemical studyTwenty antibodies were used to observe their expression at the hepatic stem cell, hematopoietic cells, cardiac cells and septum transversum mesenchyme to attain the following aims. (D Understanding the phenotypes of hepatic stem cell ? Revealling the induction of adjacent cardiac cells, septum transversummesenchyme, endothelium on hepatic stem cell. (3) Understanding the functional manner and time of those factors byobserving the temporal and spatial expression of the factors and theirreceptors.4. RT-PCR and in situ hybridizationTo understand the effect of HNF4a and FTNF6 on proliferation, differentiation of hepatic stem cell, we observed the temporal and spatial expression of FfNF4a and HNF6 mRNA of livers at various development stages of mouse.2. Results and discussion2.1 The morphological property of hepatic stem cellThe hepatocytes of 3~ 5W of gestation displayed the typical characteristic of immature cells: smaller size, a round or ovoid nuclei with dark color, scant cytoplasm with slight blue and a high ratio of nuclei/cytoplasm. They were positive for AFP and c-Met. As a result, we supposed that hepatocytes of 3~5W were homogenous and belong to thehepatic stem cells. At 6W, a part of hepatocytes became larger with slight color nuclei, which were negative for AFP and c-Met. The morphologic change of hepatocyte indicates that the hepatic stem cell began to differentiate into hepatocytes. At E10-12W, the AFP and c-Met positive cells mainly located at the periportal region. These indicated that, like oval cells in the adult liver, the hepatic stem cell located at the periportal region atE10-12W.The immunohistochemical positive reaction of CK19 became detectable at E7W, which dispersed all the liver. At El0-11W, the reaction was confined at the hepatocytes, adjacent to the portal region, ductual plate and biliary epithelial cells. At E12W, the positive reaction was only found at the ductual plate and biliary epithelial cells. It's known that all the cells of the ductual plate and biliary epithelial cells were positive for AFP, c-Met and CK19. The results implied that the cells characterized by AFP+/ c-Met+/CK 19+belong to the progenitor cells of biliary epithelial cells.2. The expression of growth factors and their receptors in the hepatic stem cell, endothelium, hematopoietic cells, primitiveheart and septum transversum mesenchyme.Hepatic stem cells of E3-10W were positive for c-Met, but negative for its ligand, HGF. Meanwhile, the cardiac cells, septum transversum mesenchyme and hematopoietic cells, expressed HGF. These results indicated that HGF produced by cardiac cells, septum transversum mesenchyme and hematopoietic cells, might combine c-Met secreted by heptocytes, and trigger the proliferation and differentiation of the hepatic stem cell.At E3-4W, the hepatic stem cell, hematopoietic cells, and endothelium were negative for IGF-I, TGF-pl, and their receptors. At E5-12W, all thegrowth factors and their receptors were expressed in the hepatic stem cell, hematopoietic cells, cardiac cells, and septum transversum mesenchyme. The results implied that IGF-I and TGF-f31 have no effect of initiation on human embryonic liver formation. After 5W, IGF-I and TGF-pl secreted by hematopoietic cells, cardiac cells, and septum transversum mesenchyme might regulate the proliferation and differentiation of hepatic stem cell by para-secretion. And IGF-I and TGF-J31 secreted by hepatic stem cell could also function by auto-secretion. VEGFA-positive reaction was found at the cardiac cells at E3-7W, at septum transversum mesenchyme at E3-12W. The receptors of VEGFA, fltl and flkl were expressed at hepatic stem cell of E4-12W. These results suggested that VEGFA produced by the cardie cells and septum transversum mesenchyme might regulate the proliferation and differentiation of hepatic stem cell by papa-selection. Moreover, the VEGFA were also expressed by hematopoietic cells during E4~12W, and, as mentioned above, its receptors fltl and flkl were expressed at hepatic stem cell. So, we supposed that proliferation and differentiation of the human embryonic hepatic stem cell depended on the VEGFA produced by hematopoietic cells. At E4-6W, a part of hepatic stem cells were also positive for VEGFA, so VEGFA could also function by auto-secretion at that time. At E4-12W, VEGFC positive-reaction was found in most of hepatocytes. At E3-7W, the cardiac cells and septum transversum mesenchyme were positive for VEGFC. Most interestingly, the endotheliums within septum transversum mesenchyme were positive for VEGFC, while those within liver were negative. The receptors of VEGFC, flt4, were present at hepatic stem cell at E4 — 12W. The results demonstrated that VEGFC produced by the cardiac cells and septum transversum mesenchyme might regulate the proliferation and differentiation by para-secretion. Meanwhile, VEGFC secretion by hepatic stem cell could function by auto-secretion.3 The expression of HNF4 a mRNA by mice liverRT-PCR studies showed that HNF4a first expressed at the time of formation of liver bud, lasted through all gestation and existed in adult liver yet. In situ hybridization showed that HNF4a is expressed by hepatic stem cells and hepatocytes. Those results demonstrated that HNF4a could regulate the formation of liver bud, trigger the differentiation of hepatic stem cell towards hepatocytes, and keep the differentiated state.4 The expression of HNF6 mRNA by mice liverRT-PCR detected expression of HNF6 mRNA in the liver at E9d, the point of liver onset. Then HNF6 mRNA disappeared transiently from the liver at El 3d, but it was appeared again at E15d. Its expression lasted to adult. In situ hybridization studies showed strong HNF6 expression in the cells of adult plate and biliary epithelial cells. These results indicated that HNF6 might play a role at the onset of liver development and the differentiation of hepatic stem cell towards biliary epithelial cells.