Liver regeneration
Authors: Nelson Fausto, Jean S. Campbell, Kimberly J. Riehle
Journal: Journal of Hepatology (2012)

In this 'Snapshot' article, we take a fresh look at the fundamentals of liver regeneration in light of recent technical innovations. While the mammalian response to partial hepatectomy (PH) has been studied for decades [1Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1253) Google Scholar, 2Michalopoulos G.K. Liver regeneration after partial hepatectomy.Am J Pathol. 2010; 176: 2-13Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar], in recent years the use of new experimental techniques has not only lead to a re-affirmation of basic principles of regeneration initially described by Nancy Bucher and others [[3]Bucher N.L. Liver regeneration: an overview.J Gastroenterol Hepatol. 1991; 6: 615-624Crossref PubMed Scopus (94) Google Scholar]; it also highlights remaining questions. PH leads to tightly synchronous rounds of replication, and given that the remaining liver is uninjured, it offers a physiological way to study both proliferative pathways in normal hepatocytes and abnormal regeneration in genetically modified mice. Liver regeneration after PH has provided a deeper understanding of mammalian cell proliferation in vivo, and may provide molecular insights into the interminable self-renewal of mature cells, a property often ascribed exclusively to stem cells. Hepatocytes appear to be cell autonomous in deciding their replication fate. This concept is based on an amalgam of work from many laboratories, and has been well illustrated by Weglarz and Sandgren [[4]Weglarz T.C. Sandgren E.P. Timing of hepatocyte entry into DNA synthesis after partial hepatectomy is cell autonomous.Proc Natl Acad Sci USA. 2000; 97: 12595-12600Crossref PubMed Scopus (75) Google Scholar]. These investigators transplanted mouse hepatocytes into a rat liver and then performed 2/3 PH. The mouse hepatocytes divided at 40 h, in contrast to the surrounding rat hepatocytes, which divided at 24 h. Each hepatocyte maintained its own proliferative cadence despite similar endocrine and paracrine influences. These findings are complemented by a recent study by Wu et al., in which sequential injections of differently labeled nucleotide analogs were used to determine the time course of hepatocytes' proliferation in different zones of the liver lobule [[5]Wu Y. Guo F. Liu J. Xiao X. Huang L. He D. Triple labeling with three thymidine analogs reveals a well-orchestrated regulation of hepatocyte proliferation during liver regeneration.Hepatol Res. 2011; 41: 1230-1239Crossref PubMed Scopus (12) Google Scholar]. They demonstrated that there are three peaks of DNA synthesis after PH, initially in zone 1, then in the mid-lobule (Fig. 1A). Interestingly, 15% of pre-existing hepatocytes never divide after PH, while 11% divide at least three times. Whether this phenomenon is due to proximity to portal nutrients, contact with specific non-parenchymal cells (NPCs), altered hemodynamics, or purely intracellular events is yet unknown. How does a hepatocyte that just has divided choose to exit the cell cycle, while its sister proceeds through another round of proliferation? Whether new hepatocytes in the regenerating liver are derived from adult hepatocytes, intrahepatic stem cells, or circulating stem cells is an ongoing controversy. Early experiments suggested the former case, but in the current era of stem cell biology, many authors have favored the hypothesis of an expansion of a progenitor cell population during regeneration and normal liver homeostasis, the so-called 'streaming liver hypothesis' [[6]Furuyama K. Kawaguchi Y. Akiyama H. Horiguchi M. Kodama S. Kuhara T. et al.Continuous cell supply from a Sox9-expressing progenitor zone in adult liver, exocrine pancreas, and intestine.Nat Genet. 2011; 43: 34-41Crossref PubMed Scopus (643) Google Scholar]. Willenbring and colleagues have recently addressed this question by developing a means of stably expressing enhanced yellow fluorescent protein (EYFP) in adult hepatocytes [[7]Malato Y. Naqvi S. Schurmann N. Ng R. Wang B. Zape J. et al.Fate tracing of mature hepatocytes in mouse liver homeostasis and regeneration.J Clin Invest. 2011; 121: 4850-4860Crossref PubMed Scopus (326) Google Scholar]. This system allowed them to 'fate map' these cells over time, and to confirm that adult hepatocytes are the source of new cells during normal liver homeostasis. Further, they found that after PH, the vast majority (∼99%) of new hepatocytes came from pre-existing adult hepatocytes, confirming that progenitor cells do not play an important role in normal liver homeostasis and regeneration. The use of these innovative techniques, in conjunction with experimental models of inflamed, fibrotic, or fatty livers undergoing resection, will further increase our understanding of compromised regeneration, as progenitor cells may play a more prominent role in restitution of hepatocyte mass during liver injury [[8]Greenbaum L. Wells G. The role of stem cells in liver repair and fibrosis.Int J Biochem Cell Biol. 2011; 43: 222-229Crossref PubMed Scopus (67) Google Scholar]. In addition to hepatocyte-autonomous signals (Fig. 1B), endocrine and paracrine factors are critical to normal regeneration, and extensive work has focused on the role of the liver microenvironment, i.e. NPCs and the extra-cellular matrix (ECM), in liver homeostasis and regeneration [1Fausto N. Campbell J.S. Riehle K.J. Liver regeneration.Hepatology. 2006; 43: S45-S53Crossref PubMed Scopus (1253) Google Scholar, 2Michalopoulos G.K. Liver regeneration after partial hepatectomy.Am J Pathol. 2010; 176: 2-13Abstract Full Text Full Text PDF PubMed Scopus (548) Google Scholar, 9Kmiec Z. Cooperation of liver cells in health and disease. In: Beck F, Christ B, Kriz W, Kummer W, et al., editors. Advances in Anatomy Embryology and Cell Biology, vol. 161. Springer; 2001.Google Scholar] and (Fig. 1C). Recent studies from Shahin Rafii's laboratory have focused on the role of endothelial cells in supporting normal hepatocyte proliferation and, more predictably, in restoring functional vasculature to the regenerating liver [[10]Ding B. Nolan D.J. Butler J.M. James D. Babazadeh A.O. Rosenwaks Z. et al.Inductive angiocrine signals from sinusoidal endothelium are required for liver regeneration.Nature. 2010; 468: 310-315Crossref PubMed Scopus (551) Google Scholar]. Other NPCs, such as Kupffer cells, stellate cells, and intrahepatic lymphocytes also provide critical signals to hepatocytes during regeneration [[8]Greenbaum L. Wells G. The role of stem cells in liver repair and fibrosis.Int J Biochem Cell Biol. 2011; 43: 222-229Crossref PubMed Scopus (67) Google Scholar], (Fig. 1B); we predict that these intercellular interactions would be even more crucial during regeneration in livers with an altered microenvironment [9Kmiec Z. Cooperation of liver cells in health and disease. In: Beck F, Christ B, Kriz W, Kummer W, et al., editors. Advances in Anatomy Embryology and Cell Biology, vol. 161. Springer; 2001.Google Scholar, 11Clavien P. Petrowsky H. DeOliveira M.L. Graf R. Strategies for safer liver surgery and partial liver transplantation.N Engl J Med. 2007; 356: 1545-1557Crossref PubMed Scopus (774) Google Scholar]. For example, do steatosis, inflammation and fibrosis shorten the self-renewal capacity of hepatocytes? Would paracrine signaling pathways override the hepatocyte cell kinetics or the indefatigable capacity to replicate? In elegant experiments aimed at addressing the role of the abnormal microenvironment in affecting hepatocyte function and proliferation, Liu et al. isolated primary hepatocytes from normal rats and from rats with compensated or decompensated cirrhosis [[12]Liu L. Yannam G.R. Nishikawa T. Yamamoto T. Basma H. Ito R. et al.The microenvironment in hepatocyte regeneration and function in rats with advanced cirrhosis.Hepatology. 2011; https://doi.org/10.1002/hep. 24815Crossref Google Scholar]. They then performed repopulation experiments with those cells, and found that hepatocytes from normal rats or compensated cirrhotics were immediately able to engraft and proliferate in the normal microenvironment of the recipient liver. Interestingly, though the hepatocytes from rats with decompensated cirrhosis initially did not expand (or produce albumin), after two months in the recipient, their function was re-established. It would be fascinating to know how these cells would respond to a proliferative stimulus at this later time point. Our hope and anticipation is that the use of innovative approaches, such as those outlined above, will lead to a deeper understanding of liver regeneration, so that therapies will soon be available for patients with acute and chronic liver failure, and will facilitate liver resection in patients who would not otherwise have been considered as surgical candidates [[11]Clavien P. Petrowsky H. DeOliveira M.L. Graf R. Strategies for safer liver surgery and partial liver transplantation.N Engl J Med. 2007; 356: 1545-1557Crossref PubMed Scopus (774) Google Scholar]. The authors declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. The underlying research reported in the study was funded by the NIH Institutes of Health. With gratitude, Drs. Campbell and Riehle wish to acknowledge the great contributions and mentorship of Dr. Fausto, who passed away during the writing of this manuscript.