Recent findings have rekindled an old debate about whether hepatocytes or liver progenitor cells are the primary source of new hepatocytes in liver homeostasis and regeneration. Hepatocytes are likely candidates because they have stem cell–like proliferative capabilities (7
), which make liver progenitor cells dispensable. However, data obtained with new mouse models (18
) and refined analyses of human livers (17
) suggest that liver progenitor cells contribute significantly, if not predominantly, to replacing hepatocytes during normal turnover and after acute injury. Other recent reports have raised the possibility of lineage conversion of hepatocytes in response to biliary injury; the data suggest that hepatocyte differentiation is not fixed, but that hepatocytes can supply new biliary epithelial cells when the regenerative capabilities of both biliary epithelial cells and liver progenitor cells are impaired (26
To test these new findings, we generated an in situ hepatocyte fate-tracing model based on rapid and specific marker gene activation in all hepatocytes of adult R26R-EYFP mice with the nontoxic vector AAV8-Ttr-Cre. We found that all newly formed hepatocytes in the normal adult liver are derived from preexisting hepatocytes. Our result contradicts the finding by Furuyama et al. that liver progenitor cells are primarily responsible for liver homeostasis (18
) and supports the previous paradigm of hepatocyte self duplication as the principal mechanism. While we can only speculate on the reason for the finding by Furuyama et al., possibly their experimental system, which was based on induction of Cre expression in liver progenitor cells and biliary epithelial cells in adult mice, caused toxicity and liver progenitor cell activation. An unrecognized environmental factor may have subsequently perpetuated liver progenitor cell activation by causing subclinical chronic hepatocyte injury. This hypothetical, but not unlikely, scenario would explain why homeostatic hepatocyte replacement occurred in their mice in the form of bridging between periportal areas, a characteristic feature of liver progenitor cell–mediated repair of chronic hepatocyte injury (18
We also used our hepatocyte fate-tracing model to identify the origin of new hepatocytes in liver regeneration. In accordance with findings by Furuyama et al. (18
), we found that liver progenitor cells contribute to hepatocyte replacement after 2/3 PH and CCl4 intoxication. This finding is surprising because hepatocytes lost due to these nontoxic or hepatocyte-specific insults were previously believed to be replaced only by self duplication of residual hepatocytes (19
). However, our results also show that the number of hepatocytes derived from liver progenitor cells after 2/3 PH or CCl4 intoxication is very small and thus most likely irrelevant. Moreover, we found that chronic, but not acute, CCl4 intoxication produces liver progenitor cell–derived hepatocytes. These results contradict the finding of Furuyama et al. that acute CCl4 intoxication leads to rapid production of a substantial number of hepatocytes by liver progenitor cells (18
). Viewed together, our findings suggest that bile duct proliferation, which is triggered by both chronic CCl4 intoxication (57
) and 2/3 PH (24
), is a prerequisite for the emergence of liver progenitor cell–derived hepatocytes.
Finally, we failed to find hepatocyte-derived biliary epithelial cells in our hepatocyte fate-tracing model. Our results obtained with BDL argue against the recently proposed concept of biliary injury causing conversion of hepatocytes into biliary epithelial cells (26
). As was done in the original studies (26
), we identified biliary epithelial cells based on CK19 expression. However, differences also exist between our experiments and the original ones that may warrant further investigation. First, we used in situ hepatocyte fate tracing in mice, whereas the original studies used hepatocyte transplantation into rats. Although unlikely, transplanted rat hepatocytes may be more amenable to lineage conversion than resident mouse hepatocytes. Second, we did not combine BDL with biliary toxins. A study in rats found that, while at least 1.75% of all biliary epithelial cells were hepatocyte derived after BDL alone, the frequency could be markedly increased by additional exposure to methylene dianiline (DAPM), a biliary toxin that blocks proliferation of biliary epithelial cells and liver progenitor cells (27
). Although little information is available on the effects of DAPM in mice (62
), combining BDL with DAPM intoxication may block proliferation of both biliary epithelial cells and liver progenitor cells in these animals as well. Nevertheless, we are confident that our system would have detected even a very small number of hepatocyte-derived biliary epithelial cells, considering that we could identify rare liver progenitor cell–derived hepatocytes after 2/3 PH or chronic CCl4 intoxication. Third, our BDL protocol entailed ligation of the left hepatic duct for 10 days, not the common bile duct for 30 days, as in the rat studies. We chose this protocol because it avoids the severe systemic effects of common BDL in C57BL/6 mice, including hepatocyte injury, weight loss, and mortality, while causing indistinguishable ductular reactions in the affected liver lobes (58
). Therefore, we cannot rule out that by avoiding injury and death of hepatocytes, we may have limited compensatory hepatocyte proliferation, which is generally viewed as a promoter of lineage conversion (32
). On the other hand, recent studies showed that proliferation is not needed for lineage conversion if the cell types are developmentally related, such as pancreatic exocrine and endocrine cells (64
), B cells and macrophages (65
), or, in this case, hepatocytes and biliary epithelial cells.
Hepatocyte-derived biliary epithelial cells were also absent in our hepatocyte fate-tracing model after DDC feeding. Because most DDC-induced liver progenitor cells express CK19 (10
), our results are consistent with previous findings that transplanted hepatocytes are not precursors of liver progenitor cells in mice (66
). Nevertheless, it is in principle possible that our analysis missed rare hepatocyte-derived liver progenitor cells that were not expressing CK19. However, if such cells were present, it would be surprising that none of them differentiated into CK19-positive biliary epithelial cells during the extensive expansion of ductular reactions triggered by 8-week-long DDC feeding.
In conclusion, our study settles a long-standing debate by demonstrating that liver homeostasis is mediated by self duplication of mature hepatocytes and does not involve liver progenitor cells. Moreover, we show that liver progenitor cells contribute only minimally to regeneration of acutely lost hepatocytes. Thus, our results support the view that liver progenitor cells provide a backup system for injury states in which the proliferative capabilities of hepatocytes are impaired. Our findings underscore the importance of hepatocytes for liver regeneration and suggest that harnessing liver progenitor cells for therapy will require knowing the signals that trigger their activation. We also used our hepatocyte fate-tracing model to test the emerging concept that hepatocytes spontaneously convert into biliary epithelial cells in states of biliary injury. We found no evidence for hepatocyte-derived biliary epithelial cells or liver progenitor cells in 2 commonly used models of biliary injury. In the future, hepatocyte fate tracing could be used to determine whether hepatocyte lineage conversion can be forced by overexpression of biliary-specific transcription factors. Hepatocyte fate tracing could also serve to advance our understanding of hepatocarcinogenesis by determining whether liver cancers exhibiting liver progenitor cell characteristics derive from dedifferentiated hepatocytes or liver progenitor cells. In addition, AAV8-Ttr-Cre could be generally useful for liver research by facilitating timed gene inactivation in all hepatocytes, but no other liver cells, of conditional knockout mice.