Isolation and Characterization of AFP+/DLK1+ Double-Positive Hepatic Stem/Progenitor Cells from Fibrotic PHx Model

Background The liver is the largest digestive organs of vertebrates and high incidence of diseases. In addition, liver also is an organ with strong regenerative capacity. At present, partial hepatectomy (PHx) is still considered the most effective treatment in various treatment strategies. After the liver is undergoing acute injury such as PHx, the remaining mature liver cells will be excessively hypertrophic, re-enter the cell cycle to start proliferation and restore liver function. In recent years, although some progress has been made in elucidating Hepatic Stem/Progenitor Cells (HSPCs) dependent liver regeneration, the isolation, characterization and the in vitro cultivation of HSPCs still pose challenges. Methods Liver fibrosis is induced in mice by subcutaneous injection of CCl ₄ in the back for 8 weeks. After that, 2/3 PHx was used to establish a fibrotic PHx model mouse, and the samples were taken at after PHx 12h, 24h, 48h, 4d and 7d. Expression of HSPCs associated markers including AFP, DLK1, KI67, and LGR5 was analyzed using quantitative real-time PCR and immunofluorescence. The HSPCs were isolated through density gradient differential centrifugation, followed by flow cytometry analysis to characterize the AFP ⁺ /DLK1 ⁺ double positive expression. Results The induction of fibrosis was successfully achieved by subcutaneously injecting 20% carbon tetrachloride(CCl ₄ ) (0.02ml/g) into the dorsal region twice a week for 8 weeks, thereby establishing a reliable fibrotic 2/3 PHx model mice. Pre-injection of CCl ₄ promotes liver regeneration after 2/3 PHx, which had a rapid stage at 24h ~ 48h. Of note, AFP ⁺ /DLK1 ⁺ double-positive cells exhibit stem cell-like properties involved in liver regeneration. Gene expression analysis revealed that the higher concentration of 50–70% layer cells when AFP ⁺ /DLK1 ⁺ double-positive cells were isolated through density gradient differential centrifugation. Interestingly, adult HSPCs and embryonic HSPCs exhibit distinct peroll layers and require different culture conditions in vitro. The quantity of adult HSPC is lower, and their cultivation presents greater challenges. Conclusion AFP and DLK1 as markers to permit selection of the HSPCs population from fibrotic PHx model. Our results demonstrated that the 48h after PHx in fibrotic liver was the most vigorous stage of liver regeneration, making it the optimal period for the isolation and extraction of HSPCs, and the utilization of density gradient differential centrifugation is a highly efficient approach for the isolation and purification of HSPCs.