Chasing the facultative liver progenitor cell
Authors: Y.Y. Dan
Journal: Hepatology (2016)

The controversy on the origins and physiological role of facultative liver progenitor cells in liver regeneration was revisited by Lu et al.1 In this elaborate study, the authors used an inducible AhCre mouse model to delete Mdm2 in hepatocytes to simulate a model of massive liver injury and hepatocyte senescence. Florid ductular proliferation of progenitor cells occurred and contributed to significant reconstitution of the liver. Complete recovery of the liver was seen in a less lethal model of AhCre+Mdm2flox/− mice with heterozygotic deficiency of Mdm2. Using markers found on these progenitor cells, fluorescence-activated cell sorting was used to isolate a similar population of progenitor cells from livers of mice given the choline-deficient, ethionine-supplemented (CDE) diet. These cells were traced to a biliary origin using tamoxifen-induced Krt19CreERTLSLtdTomato labeling and could be expanded in vitro up to 30 passages. Transplantation of these cells into the AhCre+Mdm2flox/flox model showed significant repopulation into both the hepatocytic and biliary lineages with improvement of liver histology and function. Worldwide, the mismatch between liver graft availability and number of patients waiting for liver transplantation has widened exponentially, resulting in more patients dying while on the waiting list. This unmet clinical need has spurred much effort to develop cellular transplantation as an alternative to whole-organ transplantation to correct diseases of liver insufficiency. The adult hepatocyte is the key primary cell responsible for regeneration during normal liver homeostasis as well as in most instances of clinical acute liver injury including surgical hepatectomy.2 Accumulative data over the last 30 years have led to the concept that under conditions of hepatocyte senescence, a second-line defense mechanism is triggered where normally quiescent cells residing in the terminal ductules (canals of Hering) will be activated to assume the facultative role of stem/progenitor cells. These cells are believed to proliferate and differentiate into one or more daughter lineages to regenerate the liver.2 It is worth noting that these cells are more appropriately termed “progenitor cells” as opposed to “stem cells,” which strictly, would require proof of capacity for indefinite self-renewal. Regardless, the ability to successfully isolate and expand a facultative progenitor population for meaningful repopulation and regeneration of an injured liver has become the holy grail of liver stem cell biologists. The conventional concept of the regenerative role of facultative liver progenitor cells was, however, challenged in recent years when lineage tracing experiments showed discordance in the origins of regenerating hepatocytes. Furuyama et al., using tamoxifen-induced Cre-lox labeling of the Sox-9 cholangiocyte marker, found that hepatocytes did seemingly stream from the Sox-9 expressing biliary cell.3 Among several groups who reported similar observations, Espanol-Suner et al.4 used OPN-iCre and showed that biliary progenitor cells underwent ductular proliferation and contributed up to 2.5% of hepatocytes in the CDE mouse model. In contrast, several independent groups,5, 6 using different gene tracing markers in carefully controlled experiments, came to an entirely opposite conclusion. In all their injury models, there was no physiological regenerative role for facultative progenitor cells or biliary ductular cells. Hepatocyte regeneration was entirely from hepatocytes, and the biliary ductular proliferating cells contributed minimally, if at all, to hepatocytic regeneration. To further complicate issues, Wang et al.7 very recently reported that, in normal homeostasis, it is the self-renewing Axin2-positive pericentral hepatocytes around the central vein that regenerate surrounding hepatocytes (see Fig. 1). Amid this controversy, the article by Lu et al.1 provides persuasive evidence to reaffirm the conventional concept that progenitor cells from a biliary origin did contribute significantly to regeneration, at least in a model where there was massive injury and where adult hepatocytes were rendered senescent. In this model, deletion of Mdm2 up-regulated nuclear p53 in 98% of hepatocytes and virtually all hepatocytes demonstrated p53-mediated senescence by p21 expression. Regenerated hepatocytes were nuclear p53-negative, indicating their origins in nonparenchymal cells. Blocking the ductular proliferation by manipulating the TWEAK/Fn14 pathway abrogated the recovery, lending direct support to the physiological importance of ductular proliferation in regeneration. A few possibilities may have accounted for the observations that flew against the weight of evidence that liver progenitor cells are not from the biliary lineage. Firstly, the complete senescence rendered in all the hepatocytes in this model may have shut off any hepatocytic contribution to the ductular proliferation, leaving the biliary cells as sole contributor to the ductular proliferation.6 Secondly, the extreme survival selection pressure may have created an artificial environment pushing the plasticity of biliary cells, either cholangiocytes or terminal ductular cells, into regenerating the damaged liver, something that may not occur in other models of injury. Thirdly, although hypothetical, one wonders if damaged hepatocytes could have escaped from the induced p53 labeling amid the extreme selection pressure, undergone biliary metaplasia and ductular proliferation, and repopulated the liver.6 Lineage tracing using an additional hepatocyte marker would have unequivocally answered the question of progenitor cell origins in this model but was unfortunately not incorporated. Notwithstanding the uncertainty on the origins of these cells, Lu et al.1 have kept alive the notion of using facultative progenitor cells as cellular therapy for liver regeneration. CD45−CD31−Ter119−EpCAM+ CD24+CD133+ progenitor cells could be isolated from CDE livers by fluorescence-activated cell sorting and expanded unaltered in vitro at an efficiency of 32 doublings in 10 passages. Although the techniques described to achieve this were not necessarily novel or unique, it is exciting that the logistic limitation of scaling up progenitor cell expansion for clinical use, can be overcome. More importantly, the authors demonstrated convincingly that in vitro expanded progenitor cells can result in significant repopulation of the AhCre+Mdm2flox/flox liver and register clinical benefit. This has hitherto been shown only as a principle of proof and has never reached the magnitude achieved in this study. One limitation though was that the transplantation experiments used progenitor cells from the CDE model. Given the controversy that CDE progenitor cells may be derived from hepatocytes, it would be interesting if the progenitor cells from the AhCre model or even normal uninjured liver would similarly show therapeutic efficacy in this injury model. How do we draw relevance from these experiments to the clinical setting? It appears that the liver is a highly complex organ where the plasticity of various cell types, even differentiated ones, allows different candidate cells to step forward under different experimental conditions, to become the facultative progenitor cells.6 Using the Krt19CreERTLSLtdTomato label, the authors concurred that the contribution of biliary KR19 labeled cells to regenerating hepatocytes is minimal in both normal liver homeostasis and the CDE model. It only becomes prominent in the AhCre model due to induction of complete injury and maximal senescence. Senescence of the hepatocyte is believed to be widespread in clinical liver diseases, and ductular proliferation is increased in patients with the most severe liver injury.8 However, the extreme survival selection pressure generated in Lu et al.'s model is probably unprecedented, and how relevant this is to human liver diseases is not known. From a logistic perspective, these cells will be plentiful for harvesting in recipient explanted livers, although the concern is that the genomic and epigenetic alterations sustained in these injury-induced cells may increase the risks of malignancy when these cells are transplanted. Regardless of the true origins and physiological relevance to the clinical setting, Lu et al. have shown that there are facultative progenitor cells that can be isolated, expanded, and transplanted into injury models, where they show significant regeneration and improved outcomes. That is a big affirmation for those working to make this strategy a reality. The chase is certainly on again. Y.Y. Dan, M.D., Ph.D.1-4 1Department of Medicine, Yong Loo Lin School of Medicine 2Division of Gastroenterology and Hepatology, University Medicine Cluster National University Health System 3Cancer Science Institute, National University Singapore 4Genome Institute of Singapore, Agency for Science, Technology, and Research Singapore