Triple labeling with three thymidine analogs reveals a well-orchestrated regulation of hepatocyte proliferation during liver regeneration

Aim: After a two-thirds partial hepatectomy (PHx) in rodents, the remaining cells will proliferate and restore the lost liver mass within 7days. Previous studies have proved that the residual hepatocytes proliferate in a synchronous manner. However, the existing data can not reflect the chronicle of individual hepatocytes proliferation during liver regeneration. Methods: In this study, a combination of pulse and continuous labeling using three thymidine analogs, Bromodeoxyuridine (BrdU), Chlorodeoxyuridine (CldU) and Iododeoxyuridine (IdU), were used to analyze the cell proliferation of rat liver after PHx. This strategy allows us to follow an individual cell for more than one cell cycle and to define how many cells and which cells undergo multiple divisions. Results: The residual hepatocytes clustered into three subpopulations to initiate the proliferation sequentially, and the corresponding percentage of each was 32%, 17%, and 36%. Meanwhile, the remaining 15% of hepatocytes never proliferated. In addition, the periportal hepatocytes were the first to respond to PHx stimulation and re-proliferated synchronously at 54h. Furthermore, at least 11% of residual hepatocytes were identified to divide thrice or more. Conclusion: Based on the present analysis, we concluded a sequential model of the initial proliferation in residual hepatocytes, and for the first time, quantitatively elucidated the proliferation manner of three subpopulations during liver regeneration.