Establishment And Characterization Of The Hepatic Stem Cell Line From Retrorsine-treated Mouse

The liver is an important organ in the body where protein synthesis and metabolism of exogenous and endogenous substrates are performed. Liver is very sensitive to exogenous toxin injure. In response to massive necrosis, when hepatocytes cannot respond to a growth stimulus and during some regimes of chemically induced hepatocarcinogenesis, numerous lines of evident indicate the persistence of a few "novel" cells in the periportal area of adult liver. These cells were not present in normal liver and can differentiate into hepatocytes and bile duct cells under certain physiologic or experimental conditions.Gordon GJ et al recently described the cellular responses and time course for liver regeneration after PH in rats with retrorsine-induced hepatocellular injury. This model involves the prior administration of the DNA-binding pyrrolizidine alkaloid retrorsine to rats blocking native hepatocyte proliferation. With a mitogenic stimulus such as PH, retrorsine-injured hepatocytes undergo apoptosis and are selectively removed from the hepatic parenchyma. The emergence and rapid expansion of a population of small hepatocyte-like progenitor cells (SHPCs) contribute to the restoration of live mass after PH. In the current study, we tried to isolate liver progenitor cells from retrorsine-treated mouse. After long-term culture and purification, the pure epithelial cell population was established in cobblestone fashion with high nuclear-to-cytoplasm ratios. In our culture condition, the LEPCs express oval cell markers CK 19, CK 14, OV6 and OC.10, but not OC.2 and OC.5. The cells also express c-met, the receptor for hepatocyte growth factor (HGF). Antigens traditionally associated with haematopoietic stem cells, including c-kit, Thy-1 and CD 34, can be expressed by oval cells. LEPCs express haematopoietic stem cell marker Thy-1, c-kit and CD 45, but not CD 34. The hepatocyte markers albumin and AFP, Cholangiocyte markers CK 7 and -GT, miscellaneous molecules CK 18 and CK 8 cannot be detected. The antibodies of c-kit, CD 45, OV6 and CK 19 decorated more than 90% cells. From the antigen profile expressed by the cells, the cells are of the character of hepatic stem cells, so are named liver epithelial progenitor cells (LEPCs).Sodium butyrate and DMSO are commonly used differentiation promoting agents. Upon exposure the culture cells to 5 mM sodium butyrate, the growth of LEPCs was significantly arrested. Double nuclear cells, character of hepatocyte in vivo, can be observed after four days in the culture. Ten days later, about 20% cells became double nuclear. Our ultrastructural observations provided more convincing evidence that the hepatic phenotype was induced. Before the sodium butyrate treatment, the cells contained few organelles except some mitochondria and ribosome. After exposure to sodium butyrate, the cells contained well-developed organelles such as mitochondria, lysosomes, Golgi apparatus, rough and smooth endoplasmic reticulum. Many glycogen granules scattered in the plasma. Notably, a bile canaliculus was observed in the intercellular space of adjacent two cells. Molecular markers, such as CK 19, CD 45, CK 14 and OC.10, fail to be detected in the first five days of differentiation, while the expression of OV6 and c-met last even ten days later. Hepatocyte specific markers albumin, Apo A4, Apo B, aldolase B and ADH were expressed ten day after induction, which indicated LEPCs gave rise to functional hepatocyte. The extracellular matrix plays important roles in the cell behavior in vitro and in vivo. Matrigel were widely used to induce differentiation of variable cell types. Cultured in the Matrigel, the cells aggregated together and formed tube-like structure. The distinct characteristic of hepatic stem cells is the potential to give rise to mature hepatocytes and bile duct cells. From the results mentioned above, LEPCs are of the traits of hepatic stem cells.To follow the differentiation of LEPCs in vivo, LEPCs were tagged with reported gene EGFP, DsRED and Lac Z by retroviral methods. We transplante...