Activated Hepatic Stellate Cells Promote The Dedifferentiation Of Rat Hepatocytes Into Hepatic Progenitor Cells

【Background and Objective】Hepatic progenitor cells (HPCs) are bipotential cells that can differentiate towards the biliaryand the hepatocytic lineage in damaged liver. On severe or chronic liver injury, HPCs areactivated and assumed to repair the liver damages by differentiating to mature cells. Moreover,the number of HPCs was shown to be related to both the severity of liver disease and the riskfor development of hepatocellular carcinoma. HPCs coexpress markers of biliary epithelial cells(such as OV-6, PCK and Sox9), hepatocytes (such as albumin) and hepatoblasts (such as AFPand Dlk1). Their cellular origin remains obscure. Previous evidence has suggested that HPCsmay originate from the canal of Hering. A possible contribution of hepatocytes as an origin ofHPCs can also be considered. Notably, an intriguing possibility that hepatic stellate cells (HSCs)are capable of serving as “HPCs” has also been suggested and is under continuous debate.Besides, the possibility that HPCs can derived from bone marrow stem cells has beenconsidered. These studies strongly suggest that HPCs are a heterogeneous population of cells.HSCs are located in the space of Disse between the sinusoidal endothelial cells and hepaticepithelial cells. HSCs are quiescent in the healthy liver and activated in the injuried liver. Whenactivated, HSCs transdifferentiate into myofibroblast-like cells, which produce extracellularmatrix (ECM) and secrete cytokines. It has been widely accepted that the activation of HSCs isa key event in the process of liver fibrosis. In addition, there is growing evidence suggesting thatHSCs also function to promote liver regeneration and even serve as HPCs themselves.Based on these studies, this study aimed to clarify the effect of activated HSCs on thegeneration of HPCs using animal models and to investigate the cellular origin of HPCs by co-culture system in vitro.【Methods】1. The relationship between the activation of HSCs and generation of HPCs1.1Liver tissues were collected from patients with acute hepatitis, liver fibrosis and livercirrhosis. Immunofluorescence assay was used to detect the expression of PCK and α-SMA.1.22-Acetylaminofluorene (2-AAF)/partial hepatectomy (PH) model was established toinduce HPCs in Sprague-Dawley (SD) rats. To establish this model, the rats were treated withintragastric2-AAF (10mg/kg) administration once a day for14days. On Day6, the ratsreceived a70%partial hepatectomy without the2-AAF administration. On Day0,4,6and9post-PH, the rats were sacrificed. The expression of PCK and α-SMA was detected byimmunofluorescence assay.1.3N-acetyl-paraaminophen (APAP) model was established by treating SD rats with onedose of APAP (300mg/kg) intraperitoneally. The rats were sacrificed at24h after APAPtreatment. Liver tissues were collected to detect the expression of PCK and α-SMA byimmunofluorescence assay.2. The effect of activated HSCs on the generation of HPCs2.1The effect of GdCl3and Gliotoxin on Kuppfer cells and activated HSCs2.1.1SD rats were injected with20%Carbon tetrachloride (CCl4)(2ml/kg)intraperitoneally once every other day for two times, and they were injected with one dose ofGdCl3(10mg/kg) via tail vein at48h after the last CCl4injection. Liver tissues were collectedat24h after GdCl3injection. The expression of CD68and α-SMA was detected byimmunofluorescence assay.2.1.2SD rats were injected with two doses of20%CCl4(2ml/kg) intraperitoneally onceevery other day, and were injected with one dose of Gliotoxin (3mg/kg) intraperitoneally at48h after the last CCl4injection. Liver tissues were collected at24h after Gliotoxin injection.The expression of CD68and α-SMA was detected by immunofluorescence assay. 2.2The effect of inhibiting HSCs activation on the generation of HPCs2.2.1Primary SD rat HSCs were isolated and cultured in DMEM medium in vitro. Culturesupernatant of cells on day3(quiescent HSCs) and day12(activated HSCs) was collected.2.2.2On the day before PH in2-AAF/PH model or the injection of APAP, SD rats weretreated with GdCl3to reduce Kuppfer cells and inhibit HSCs activation. Liver tissues werecollected at72h after PH or injection of APAP. The expression of PCK and α-SMA wasdetected by immunofluorescence assay to clarify the effect of inhibiting HSCs activation onthe generation of HPCs.2.2.3In the same model as mentioned above, after treated with GdCl3to inhibit HSCsactivation, the rats were injected intraperitoneally with4ml culture supernatant from quiescentHSCs or activated HSCs one time at24h after PH or APAP injection. The expression of PCKand α-SMA was detected by immunofluorescence assay and comparative study was made.2.3The effect of eliminating activated HSCs on the generation of HPCs2.3.1In2-AAF/PH model,20%CCl4(2ml/kg) was injected intraperitoneally to induceHSCs activation on day1,3and the SD rats were treated with Gliotoxin on day5. Livertissues were collected at72h after PH and the expression of PCK and α-SMA was detected byimmunofluorescence assay. Comparative study was made.2.3.2In the same model as mentioned above, after the elimination of activated HSCs byGliotoxin, the SD rats were treated with4ml culture supernatant of HSCs (at different states)one time at24h after PH. The expression of PCK and α-SMA was detected byimmunofluorescence assay and comparative study was performed.3. Investigation of whether activated HSCs could promote the dedifferentiation of rathepatocytes into HPCs in vitro3.1Primary SD rat HSCs and hepatocytes were isolated. Hepatoctes were cocultured withactivated HSCs or quiescent HSCs. Morphological changes of hepatocytes were observedunder a microscope. The expression of HNF4α, PCK and Sox9was detected byimmunofluorescence assay on day0,3,5.3.2To prove the PCK (+) cells achieved from hepatocytes (cocultured with activated HSCs)were bipotential HPCs, they were cultured in biliary epithelial cells-inducing medium and hepatocytes-inducing mudium separately. The expression of PCK, CK19and Alb wasdetected by immunofluorescence assay.【Results】1. Aactivated HSCs promote the generation of HPCs1.1The generation of HPCs is closely related to the activation of HSCs1.1.1Immunofluorescence results showed that PCK (+) cells were only found around α-SMA(+) cells in human diseased liver samples.1.1.2In2-AAF/PH model, Immunofluorescence results revealed that with the increase ofdays after PH, there were more PCK (+) cells and α-SMA (+) cells. In addition, PCK (+) cellswere adjacent to α-SMA (+) cells.1.1.3In APAP model, it was demonstrated by immunofluorescence that PCK (+) cells weresurrounded by a large number of α-SMA (+) cells, indicating the close relationship between theactivation of HSCs and the generation of HPCs.1.2Activated HSCs advance the generation of HPCs1.2.1Immunofluorescence results showed that GdCl3could reduce the number of CD68(+)cells and α-SMA (+) cells. Moreover, Gliotoxin could reduce the number of α-SMA (+) cellsbut had no effect on the number of CD68(+) cells.1.2.2In2-AAF/PH and APAP models, the number of PCK (+) cells and α-SMA (+) cellswas reduced significantly after the inhibition of Kupffer cells by GdCl3. When supplementedwith the culture supernatant of quiescent HSCs, the liver didn’t display an increase in thenumber of PCK (+) cells and α-SMA (+) cells, while the number of PCK (+) cells and α-SMA(+) cells increased upon the injection of culture supernatant of activated HSCs.1.2.3In2-AAF/PH model, there were only a few PCK (+) cells and α-SMA (+) cells aftereliminating activated HSCs by Gliotoxin. When supplemented with the culture supernatant ofquiescent HSCs, the liver didn’t display an increase in the number of PCK (+) cells and α-SMA(+) cells, while the number of PCK (+) cells and α-SMA (+) cells increased upon the injectionof culture supernatant of activated HSCs. 2. Activated HSCs promote the dedifferentiation of rat hepatocytes into HPCs in vitro2.1Immunofluorescence results showed that there were PCK (+) cells in rat hepatocytescocultured with activated HSCs5days but there were no PCK (+) cells when hepatocytes werecocultured with quiescent HSCs or cultured alone. On day0, HNF4α (+) cells made up a largeproportion of hepatocytes cocultured with activated HSCs, while there were few HNF4α (+)cells on day5. In contrast, the number of Sox9(+) cells exhibited reverse change. In otherwords, on day0hepatocytes were cocultured with activated HSCs, Sox9(+) cells accounted fora very small percentage, while on day5, almost all the cells expessed Sox9.2.2The PCK (+) cells could be induced bilaterally both to biliary epithelial cells as confirmedby CK19detection and to hepatocytes as confirmed by Alb detection.【Conclusions】1. The generation of HPCs is accompanied by the activation of HSCs.2. Activated HSCs promote the generation of HPCs.3. Activated HSCs promote the dedifferentiation of rat hepatocytes into HPCs.4. HPCs arised from hepatocytes can re-differentiate towards biliary epithelial cells andhepatocytes.