Assignment topic: Liver Regeneration

Submitted To : Dr.Samina By: Razia Saleem Quaid - e - Azam University Dept : Animal Sciences MSc 2nd Semester

Index Page # 1. Introduction 3 2. Structure and functions of liver 3,4 3. Liver Regeneration 5 4. Two layers of defense against liver injury 5 5. Dynamics of liver regeneration 6 6. Stimuli of hepatic regeneration 7 7. Regeneration by hepatocytes (1st line of defense) 8 8. Signaling networks activated after partial hepatectomy 9 9. Matrix metalloproteinase family 10 10. Recent additions 12 11. Regeneration by liver progenitor cells (2nd line of defense) 13 12. Identifying the intrinsic liver stem cells 15 13. Physiological role of progenitor cells 16 14. Liver stem cell niche 17 15. Conclusion 18 16. Abbreviations 19 17. References 20

Introduction:
The liver is found in all vertebrates, and is typically the largest visceral organ. Its form varies considerably in different species, and is largely determined by the shape and arrangement of the surrounding organs. Nonetheless, in most species it is divided into right and left lobes; exceptions to this general rule include snakes, where the shape of the body necessitates a simple cigar-like form. The internal structure of the liver is broadly similar in all vertebrates.
An organ sometimes referred to as a liver is found associated with the digestive tract of the primitive chordate Amphioxus. However, this is an enzyme secreting gland, not a metabolic organ, and it is unclear how truly homologous it is to the vertebrate liver.
In human beings:
The liver is the largest glandular organ of the body. It weighs about 3 lb (1.36 kg). It is reddish brown in color and is divided into four lobes of unequal size and shape. The liver lies on the right side of the abdominal cavity beneath the diaphragm. Blood is carried to the liver via two large vessels called the hepatic artery and the portal vein. The heptic artery carries oxygen-rich blood from the aorta (a major vessel in the heart). The portal vein carries blood containing digested food from the small intestine. These blood vessels subdivide in the liver repeatedly, terminating in very small capillaries. Each capillary leads to a lobule. Liver tissue is composed of thousands of lobules, and each lobule is made up of hepatic cells, the basic metabolic cells of the liver.
Structure :
Lobes of liver :
Traditional gross anatomy divided the liver into four lobes based on surface features. The falciform ligament is visible on the front (anterior side) of the liver. This divides the liver into a left anatomical lobe, and a right anatomical lobe.
If the liver is flipped over, to look at it from behind (the visceral surface), there are two additional lobes between the right and left. These are the caudate lobe (the more superior) and the quadrate lobe (the more inferior).
From behind, the lobes are divided up by the ligamentum venosum and ligamentum teres (anything left of these is the left lobe), the transverse fissur (or porta hepatis) divides the caudate from the quadrate lobe, and the right sagittal fossa, which the inferior vena cava runs over, separates these two lobes from the right lobe.
Each of the lobes is made up of lobules; a vein goes from the centre, which then joins to the hepatic vein to carry blood out from the liver.
Cell types present in liver:
Two major types of cells populate the liver lobes: parenchymal and non-parenchymal cells. 80% of the liver volume is occupied by parenchymal cells commonly referred to as hepatocytes. Non-parenchymal cells constitute 40% of the total number of liver cells but only 6.5% of its volume. Sinusoidal endothelial cells, kupffer cells and hepatic stellate cells are some of the non-parenchymal cells that line the hepatic sinusoid.

Functions:
The liver is thought to be responsible for up to 500 separate functions, usually in combination with other systems and organs. The various functions of the liver are carried out by the liver cells or hepatocytes.
1. It synthesizes a large part of amino acids.
2. The liver performs several roles in carbohydrate metabolism.
3. The liver is responsible for the mainstay of protein metabolism, synthesis as well as degradation. 4. The liver also performs several roles in lipid metabolism: * Cholesterol synthesis * Lipogenesis, the production of triglycerides (fats). * A bulk of the lipoproteins are synthesized in the liver.
5. The liver produces coagulation factors I (fibrinogen), II (prothrombin), V, VII, IX, X and XI, as well as protein C, protein S and antithrombin.
6. In the first trimester fetus, the liver is the main site of red blood cell production. By the 32nd week of gestation, the bone marrow has almost completely taken over that task.
7. The liver produces and excretes bile (a yellowish liquid) required for emulsifying fats. Some of the bile drains directly into the duodenum, and some is stored in the gallbladder.
8. The liver also produces insulin-like growth factor 1 (IGF-1), a polypeptide protein hormone that plays an important role in childhood growth and continues to have anabolic effects in adults.
9. The liver is a major site of thrombopoietin production.

Liver Regeneration:

Liver is an interesting organ with high regenerative capacity and complex functions . Liver receives all exiting circulation from the small and most of the large intestine, as well as spleen and pancreas, through the portal vein. Its “strategic” location in relation to the food supply via the portal vein, and the unique gene-and protein-expression patterns of hepatocytes (the main functional cells of the liver) allow it to function as a biochemical defense against toxic chemicals entering through the food and as a re-processor of absorbed food ingredients. Nutrients entering the liver are transformed into secreted proteins (albumin, most coagulation factors, several plasma carrier proteins etc. in the peripheral blood), lipids sent as lipoproteins into the other tissues, carbohydrates stored in the liver as glycogen (the main glucose reserve used for stabilization of glucose levels in the blood). Synthesis of bile is essential for absorption of fat and lipophilic nutrients. As a major regulator of plasma glucose and ammonia levels, liver is essential for optimal function of the brain. Loss of liver function leads to chronic “hepatic encephalopathy” and eventually coma. The wide array of functions performed by liver towards the rest of the body has been safeguarded by evolutionary events which imparted to liver a phenomenal capacity to regenerate. This process allows liver to recover lost mass without jeopardizing viability of the entire organism. The phenomenon of liver regeneration following loss of liver mass is seen in all vertebrate organisms, from humans to fish. It is also triggered when livers from small animals (e.g., dogs) are transplanted to large recipients of the same species. The two layers of defense against liver injury :
It is now well accepted that there are two physiological forms of regeneration in the liver as responses to different types of liver injury (Fig. 1). At the frontline of defense are mature, normally quiescent adult hepatocytes, and in the majority of liver injuries due to drugs, toxins, resection, or acute viral diseases, hepatocytes are the main cell type to proliferate and regenerate the liver. The second layer of defense lies in the reserve progenitor cell population, which is also a quiescent compartment in the liver, but is activated when injury is severe, or when the mature hepatocytes can no longer regenerate the liver due to senescence or arrest.

Fig 1.

The Dynamics of Liver Regeneration :
Partial hepatectomy leads to proliferation of all populations of cells within the liver, including hepatocytes, biliary epithelial cells and endothelial cells. DNA synthesis is initiated in these cells within 10 to 12 hours after surgery and essentially ceases in about 3 days. Cellular proliferation begins in the periportal region (i.e. around the portal triads) and proceeds toward the centers of lobules. Proliferating hepatocytes initially form clumps, and clumps are soon transformed into classical plates. Similarly, proliferating endothelial cells develop into the type of fenestrated cells typical of those seen in sinusoids.
It appears that hepatocytes have a practically unlimited capacity for proliferation, with full regeneration observed after as many as 12 sequential partial hepatectomies. Clearly the hepatocyte is not a terminally differentiated cell.
Changes in gene expression associated with regeneration are observed within minutes of hepatic resection. An array of transcription factors (NF-kB, STAT3, fos and jun) are rapidly induced and probably participate in orchestrating expression of a group of hepatic mitogens. Proliferating hepatocytes appear to at least partially revert to a fetal phenotype and express markers such as alpha-fetoprotein. Despite what appears to be a massive commitment to proliferation, the regenerating hepatocytes continue to conduct their normal metabolic duties for the host such as support of glucose metabolism.
Stimuli of Hepatic Regeneration :
Hepatic regeneration is triggered by the appearance of circulating mitogenic factors. This conclusion was originally supported by experiments demonstrating that quiescent fragments of liver that had been transplanted to extrahepatic sites would begin to proliferate soon after partial hepatectomy, and also that hepatectomy in one of a pair of parabiotic rats led to hepatic proliferation in the other of the pair.
As might be expected, liver regeneration seems to be supported by a group of mitogens and growth factors acting in concert on several cell types. Some of the major and well-studied players that act together in this process include: * Hepatocyte growth factor (scatter factor) levels rise to high levels soon after partial hepatectomy. This is the only factor tested that acts by itself as a potent mitogen for isolated hepatocytes cultured in vitro. This factor is also of critical importance in development of the liver, as target deletions of its gene lead to fetal death due to hepatic insufficiency. * TNF-alpha, which stimulates proliferation of hepatic endothelial cells. * Interleukin-6, which acts as a biliary epithelial mitogen. * Epidermal growth factor. * Norepinephrine potentiates the mitogenic activity of EGF and HGF. * Insulin is required for regeneration but appears to play a permissive rather than mitogenic role.
The processes and signals involved in shutting down the regenerative response are less well studied than those that stimulate it. TGF-beta1, which is known to inhibit proliferative responses in hepatocytes, is one cytokine involved in this process, but undoubtedly several others participate

Regeneration by hepatocytes(1st line of defense) :

Regeneration of the liver after resection is actually compensatory hyperplasia rather than a true restoration of the liver's original gross anatomy and architecture. A particularly fascinating point about this process is that the degree of hyperplasia is precisely controlled by the metabolic needs of the organism, such that the process stops once an appropriate liver to body weight ratio is achieved. Two-thirds partial hepatectomy (PH) in rodents has been used extensively to study molecular and cellular mechanisms behind liver regeneration, with initial physiologic principles outlined in rats through the pioneering work of Nancy Bucher. Later, the advent of genetically modified mice allowed the study of various specific molecules and dissection of pathways implicated in regeneration. More recently, studies of global gene expression profiling have returned our thoughts to the “big picture”, as there are clearly multiple overlapping redundant pathways working in concert to achieve this impressive physiologic accomplishment.
PH is reproducible and leads to a proliferative stimulus that is initiated by an inflammatory stimulus, in the absence of significant cell death. Regeneration of the liver is critical to the survival of mammals and is therefore evolutionarily conserved. Thus, pathways leading to its completion are (with few exceptions) redundant. The phenotype of most genetically modified mouse models studied using the PH model thus consists of a delay rather than a complete abrogation of regeneration. Fig2
Fig 2 : Signaling interactions between different hepatic cell types during liver regeneration.

Signaling networks activated after partial hepatectomy :

Given the extent of cell proliferation needed to restore original mass after 2/3 PH, it is intuitive that virtually all cellular machinery be activated during regeneration, and that this could realistically entail hundreds of pathways. It is proposed that there is an initial activation of the cytokine cascade in Kupffer cells, which then stimulates growth factor and metabolic pathways in hepatocytes. Other non-parenchymal cells (stellate cells, vascular and biliary endothelial cells) proliferate after hepatocytes, presumably responding to yet another set of signals.
A great deal of recent work has focused on how pattern recognition receptors and a variety of inflammatory molecules are activated and initiate the cytokine signaling cascade after PH. As they have been extensively discussed elsewhere, we will not go into great detail about these pathways in this review. In brief, involved pathways include (at least) the activation of nuclear factor-kappa B (NF-κB) in Kupffer cells via tumor necrosis factor (TNF), lymphotoxin (from T cells), MyD88, and/or complement components, with downstream secretion of interleukin-6 (IL6). In turn, IL-6 binds its receptor on hepatocytes and leads to activation of the transcription factor signal transducer and activator of transcription 3 (STAT3). Fascinating newer work in mice with a hepatocyte-specific deletion of inhibitor-of-kappaB-kinase 2 (IKK2), which normally acts to activate NF-κB, demonstrated earlier and increased NF-κB activation in Kupffer cells, which had intact IKK2, with a concomitant decrease in NF-κB activation in hepatocytes. These animals had more rapid hepatocyte proliferation than control littermates, perhaps via prolonged JNK activation, highlighting both the cross talk between different cell types during liver regeneration and the critical importance of inflammatory stimuli in priming hepatocytes for replication.
After cytokines have triggered the G0 to G1 transition, several secondary signals then stimulate progression through the cell cycle. These growth factors are numerous and redundant to a great extent, again highlighting the physiologic importance of liver regeneration to the survival of the animal. Ligands of the epidermal growth factor (EGF) receptor have been extensively studied, including EGF itself,transforming growth factor alpha (TGFα), amphiregulin, and heparin binding EGF-like growth factor (HB-EGF). HB-EGF appears to be particularly required for a robust proliferative response, as it is differentially regulated after 2/3 versus 1/3 PH (the latter leads to minimal DNA replication). More recently, genetic loss of the EGFR itself has been investigated, either by RNA interference or constituitive deletion in mice, confirming a critical role of the signaling pathway in regeneration.

Hepatocyte growth factor (HGF) is another key hepatic mitogen active following PH. It is released from the extracellular matrix following PH to bind its receptor, c-Met, on the surface of hepatocytes. Conditional deletion of c-Met in the livers of mice was initially shown to cause either a significant delay in cell cycle entry after PH, or an inability to survive the procedure. Studies using RNAi against HGF or c-Met in rats supported the former study, showing a suppression of cell proliferation with successful knockdown of this pathway. Newer work has demonstrated that the mitogenic pathways activated via the EGFR and HGF/Met pathways might compensate for one another, as further characterization of the regenerative defect in hepatocyte-specific Met KO mice demonstrated that this defect could be partially reversed in culture by treatment of the cells with EGF. Similarly, in a study in Michelopoulos and colleagues using rats treated with RNAi against the EGFR, the resultant defect cell proliferation after PH was associated with a compensatory up-regulation of Met.

Matrix metalloproteinase (MMP) family : A family of proteins that appears to function across signaling networks is the matrix metalloproteinase (MMP) family. Through studies of animals genetically modified to lack inhibitors of MMPs (tissue inhibitors of MMPs, or TIMPs), MMPs have been shown to be important in the cleavage and release of growth factors from the extracellular matrix. Specifically, TIMP1 loss of function leads to increased MMP activity after PH, with increases in HGF activity and accelerated cell proliferation. Accordingly, a gain of TIMP1 function lead to a delay in cell proliferation. Loss of TIMP3 leads to a particularly interesting phenotype, with sustained TNF activity and ultimate hepatocyte death and liver failure. The remarkable finding was attributed to Timp3's function in inhibiting TACE. Thus, it is not just signaling pathways within the hepatocyte that are critical to regeneration; the surrounding environment is also important.
Fig 3:

The metabolic challenges facing the regenerating liver are quite impressive. The liver must continue to regulate systemic energy levels while meeting its own demands for significant nucleotide and protein synthesis needed for cell division. In fact, some of the most profound phenotypes seen in genetically-modified mice after PH have been demonstrated in those with defects in the phosphoinositide-3 kinase (PI3K) pathway. For instance, liver-specific deletion of phosphoinositide dependent protein kinase 1 (Pdk1) leads to a near-complete failure of regeneration after PH in mice. Important downstream effectors of this pathway include Akt, which activates mTOR and appears to affect cell size specifically, and p70 S6 kinase, which regulates the 40S ribosomal protein S6 to control protein synthesis and cell proliferation. Additionally, deletion of a downstream effector of mTOR, S6 protein itself, lead to a profound deficit in DNA replication after PH with specific effects on cyclin E induction. While mTOR may play a critical role in regulating cell size in response to the metabolic demands of the remaining functional hepatocytes, further characterization of how this interplay leads to initiation and termination of liver restoration after PH is warranted.
The Wnt/beta-catenin pathway has been extensively studied in a myriad of developmental processes in a variety of organs; liver regeneration is no exception. Using reporter mice, some investigators have demonstrated activation of this pathway after PH, while others have suggested that the canonical Wnt pathway is preferentially activated during the proliferation of oval cells (a type of progenitor cell). Hepatocyte specific beta-catenin KO mice regenerate in a delayed fashion after PH, however, perhaps via decreased activation of the EGFR.39 Of additional interest is the finding that constituitive over-expression of beta-catenin via an activating mutation at serine 45 leads to an acceleration of regeneration after PH and earlier development of HCC after diethylnitrosamine (DEN) injection.
While the cytokine, growth factor, and metabolic signaling networks are each vital to normal liver regeneration, significant cross talk between networks adds another level of complexity to this process. Suppressors of cytokine signaling (SOCS) are important mediators of this type of interaction, as their expression is induced by cytokines and their function is to act in a negative feedback loop to inhibit signaling through a whole host of receptors, including those of insulin and several growth factors. Specifically in hepatocytes, SOCS3 is highly induced after PH, is critical to shutting down cytokine signaling after PH and hepatocytes without SOCS3 were hyper-proliferative in response to growth factors in culture. Mice without SOCS3 in hepatocytes demonstrated enhanced regeneration after PH, and an earlier development of HCC after DEN injection, suggesting that this protein is critical in controlling normal and abnormal proliferative responses in the liver. Global regulation of transcription during liver regeneration.
Given the simultaneous activation of multiple diverse pathways that occurs after PH, one might expect significant changes in global gene expression during this process. In evaluating gene expression profiles during early G1, late G1, and the S phase of the cell cycle after PH, Greenbaum and colleagues described an initial decrease in the expression of genes involved in steroid and lipid metabolism and hormone biosynthesis, i.e. normal activities of the quiescent liver. As expected, later in G1 genes involved in protein synthesis and cytoskeletal organization were up-regulated, a pattern which continued through S phase, when expression of nucleotide metabolism genes became more prominent. Gene expression profiling was recently used to examine the differential proliferative response that occurs after 1/3 (minimal proliferation) versus 2/3 PH (robust proliferation). It was found that even 1/3 PH leads to significant changes in gene expression. Interestingly though, between 4 and 12 h after the two operations, a transcriptional shift seemed to occur, committing hepatocytes toward replication. This transcriptional shift consisted of the activation of genes enriched in transcription regulatory elements for FOXD3, FOXI1, CUX1, ER and E2F-1 at 4 h after 2/3 PH, and their replacement at 12 h by genes enriched in TREs for c-jun, CCAAT box, Myb, Ets-1, Elk-1 and USF, which are associated with DNA replication. These data demonstrate that the liver initially responds to PH with massive changes in gene expression, even if the operation does not result in DNA replication, and suggest that genomic and epigenomic changes function as a “wake up” call for quiescent hepatocytes to prepare them for the decision to replicate, which occurs 12 h after PH or later.
Micro RNAs appear to serve as an additional layer of regulation during liver regeneration. These small non-coding RNAs modulate translation by binding to specific mRNAs and either directly inhibiting their translation, or inducing degradation of those same mRNAs.While this is a relatively new area of study, initial investigations demonstrated that mice with deficient microRNA processing had a delay in the G1 to S transition after PH. In particular, miR21 is induced after PH, with repression of miR378, though the precise mRNAs that are modulated by these miRNAs have not been clearly defined. Recent additions :

Using PH in conjunction with a transplantation model and in vitro work, it was discovered that hepatocytes undergo multiple changes in ploidy during this physiologic process, perhaps predisposing to oncogenesis if aneuploid cells are allowed to continue proliferate. Additionally, further work in genetically modified mouse models has lead to the discovery of novel and at times unexpected factors that drive hepatocyte proliferation after resection. One such development was the description of the critical role of platelets and platelet-derived serotonin in liver regeneration. In particular, these investigations demonstrated that thrombocytopenic mice (or mice with a variety of functional platelet defects) had a significant impairment in hepatocyte proliferation after PH. This deficit could be corrected by reconstituting the organism's supply of serotonin, a hormone typically carried by platelets.
Mice with hepatocyte-specific over-expression of glypican 3 exhibit decreased cell proliferation and restoration of liver weight after PH.Other recent work has focused on the role of the extracellular matrix in determining the appropriate size of the liver at the completion of regeneration, i.e. regulating the termination phase of regeneration. Mice with a hepatocyte-specific loss of integrin-linked kinase subjected to PH, were left with livers an average of 58% larger than their original weights. The proposed mechanism was sustained activation of the HGF and beta-catenin pathways.
Regeneration by liver progenitor cells(2nd line of defense):

When hepatocytes are prevented from proliferating, liver progenitor cells serve as the second line of defense against liver failure. Farber first described the presence of a liver progenitor cell population in 1956 when he noted the presence of small cells with high nuclear-cytoplasmic ratio and called them “oval cells “.These cells were activated in animal models of liver injury and had bipotential ability to differentiate into hepatocytes and bile duct cells. Most of the data on this cell population has come from animal models that use toxins to inhibit native hepatocytes, in conjunction with a trigger to stimulate liver regeneration.
The adult human equivalent of these progenitor cells have been localized to the terminal bile ductules, known as the canals of Hering.This quiescent cell population acts as reserve population to be activated only when the adult hepatocytes are not able to repair and regenerate the injured liver, either due to senescence or cell cycle arrest due to liver toxins such as alcohol. Upon activation, these progenitor cells proliferate in the portal zone and are seen as a collection of progenitor cells and cells of intermediate differentiation.The “streaming liver hypothesis” proposes that these cells then migrate toward the central vein in the liver lobules as progressively differentiated daughter hepatocytes. Using mitochondrial DNA mutation tracking, It may also be possible that the liver has a multi-tiered system of regeneration. There maybe up to four potential stem cell niches, in the canal of Hering, intralobular bile ducts, periductal mononuclear cells and peribiliary hepatocytes, respectivelythis was demonstrable in normal human liver as well as in regenerative nodules of liver cirrhosis.
Fig 4
Liver progenitor cells (LPC) expansion. (a) Representative pictures of cytokeratin 19 (CK19) immunostaining: in untreated (CTL) liver, 2-acetaminofluorene (AAF)/sham liver, partial hepatectomy (PH) liver 14 days post-PH, and in AAF/PH livers 7, 10 and 14 days post-PH. Magnification × 100 (PT=portal tract, CV=central vein). (b) Graph representing morphometrical analysis of CK19-stained sections of CTL livers (n=3), AAF/sham livers (n=3), PH livers 14 days post-PH (n=4), and AAF/PH livers 7 (n=8), 10 (n=3) and 14 (n=15) days post-PH. Note the large interindividual variability in CK19+ cell expansion at each time point. (c) Graph showing CK19 mRNA levels analyzed in the same groups. P-values are for comparison with PH group 14 days post-PH. P-values <0.05 were considered as statistically significant.

.Identifying the intrinsic liver stem cell :

It is likely that the liver progenitor population is a heterogeneous group of cells, which, depending on the model from which these progenitor cells are derived, specific culture techniques, and whether the cultures are clonal, may have a different cell signature.
In humans, recent reports from several labs have identified a seemingly common progenitor cell population.In acute and chronically injured livers, as well as in developing fetal livers, these cells give rise to transit amplifying cells analogous to fetal hepatoblasts, which mature to form hepatocytes and bile duct cells. Collectively, they comprise the most well characterized entity representing the facultative human liver progenitor cell.

Fig 5 : Cluster of fetal liver progenitors.
The observation that liver progenitor cells have mixed epithelial and mesenchymal markers and the ease by which mesenchymal stem cells can be converted to hepatocyte-like cells raised the possibility that they may arise from mesenchymal lineage via mesenchymal to epithelial transition.It was proposed that progenitor cells may be derived from hepatic stellate cells, and that the sonic hedgehog pathway regulated this process. In a follow up study cell fate mapping was used to show that stellate cells could became oval cells when activated in liver injury, and that these cells participate in ductular proliferation. The notion that there is a common schema within the stellate cell driving both fibrosis and regeneration by fluxing between epithelial and mesenchymal phenotypes is an attractive one, but has not been borne out by other investigations. Careful fate mapping studies failed to show any evidence of mesenchymal to epithelial transition or vice versa during liver injury .
In light of conflicting evidence, the role of epithelial-mesenchymal transition and vice versa in liver injury and repair remains highly controversial. Nevertheless, taken in context with current evidence, it is likely that the majority of liver progenitor cells are in situ cells that are descendants of the fetal ductal plate. The main strategy in attempting to augment regeneration in the clinical setting thus lies in increasing the numbers of these progenitor cells following liver injury, either by stimulating the stem cell niche to proliferate, or simply by transplanting more progenitor cells into the injured liver.

The physiological role of progenitor cells in liver regeneration:
The role of progenitor cell regeneration in normal liver physiology is still debated.These cells likely have no significant role in day-to-day liver turnover. The progenitor compartment is activated only in severe liver injury, and the belief that it plays an important role in regenerating the injured liver comes from three lines of evidence.
First, progenitor cells are present in advanced stages of many human liver diseases in which native hepatocytes are believed to be senescent or inhibited from proliferating, such as alcoholic and non-alcoholic cirrhosis, chronic viral hepatitis, and primary biliary cirrhosis. The presence of these cells directly correlates with both inflammation and the degree of liver injury; patients with higher MELD scores appear to have more progenitor cell activation. Second, studies of chronic viral hepatitis in human patients showed that these progenitor cells are indeed surrounded by hepatocyte-like cells of intermediate differentiation, suggesting ongoing regeneration. Tracing of thymidine labeling in animal models shows that progenitor cells differentiate into both hepatocytes and cholangiocytes. Lastly, transplantation of ex-vivo progenitor cells in animal models of liver injury has been convincingly shown to engraft and repopulate the liver, further underlining the capacity for these cells to regenerate.
Interestingly, although ductular proliferation is also seen after bile duct ligation and in primary biliary cirrhosis, the response in these systems is believed to come from cholangiocytes rather than progenitor cells. In advanced primary biliary cirrhosis, when cholangiocyte proliferation is arrested, proliferating ductal cells lean towards an undifferentiated pre-cholangiocytic phenotype, suggesting that the progenitor response is tailored and specific to the injury process(98, 99, 107).
In acute liver failure, progenitor cell proliferation has also been noted as a response mechanism, which fits with the understanding that progenitor proliferation kicks in when the liver is in “dire straits”(103). A threshold of loss of 50% of hepatocytes in conjunction with reduced proliferative activity of remaining mature hepatocytes triggers the progenitor population within the first week, with appearance of intermediate hepatocytes only after that week. The degree of progenitor cell activation correlates positively with clinical outcomes.
Despite the accumulating evidence of progenitor cell proliferation in liver injury, the extent to which progenitor cell regeneration contributes to repair and the natural history of human liver disease is not known. The triggers that activate this reserve component are also not well understood. Recent evidence using mitochondrial mutation tracking suggests that some of the regenerative nodules in liver cirrhosis are clonal and are likely to have arisen from a related facultative progenitor cell from a neighboring ductular reaction(61). It is likely that this regenerative process keeps the patient compensated and delays the onset of liver insufficiency, with clinical disease occurring only when the regeneration of these cells can no longer keep up with the injury process. Yet the fact that these cells are activated to a large degree only in end stage cirrhosis or fulminant liver failure, once liver injury is not reversible, suggests that manifestation of clinical disease may be more complex than just hepatocyte insufficiency alone. If this were the case, it would limit the ability of a progenitor cell transplant to reverse clinical outcomes in such late stage disease.

The liver stem cell niche:

A stem cell environment, or “niche”, is believed to maintain the liver progenitor cell in its native state, and allows for regulatory signals to activate it when required. The companion supportive cells in this niche have long been suspected to be mesenchymal cells, such as portal fibroblasts, hepatic stellate cells or vascular endothelial cells. Yovchev et al reported that these cells are CD90 positive, explaining the previous misinterpretation of CD90 as a stem cell marker. More recent in vitro work suggests that angioblasts, CD133 or CD117 cells co-expressing vascular endothelial growth factor receptor 2 (VEGF R2), maintain and encourage the proliferation of progenitor cells in their native state. Other cell types, such as endothelial and hepatic stellate cells, support their differentiation into different lineages.
Multiple autocrine and paracrine factors have been reported to activate liver progenitor cells, and have been discussed in detail in excellent recent reviews. These include inflammatory cytokines, which are similar to those that stimulate mature hepatocyte proliferation and include the IL6 family, IL18, TNFα, interferon α and γ, stem cell factor, stromal derived factor (SDF-1), lymphotoxin beta, TNF-like weak inducer of apoptosis (TWEAK) and even the sympathetic nervous system. More recent discoveries include regulatory proteins such as MERLIN, which acts on the EGFR to regulate progenitor cell proliferation; Foxl1, a mesenchymal forkhead winged helix factor that may come from surrounding portal fibroblasts, and the Wnt/sonic hedgehog pathways that trigger ductal proliferation in alcoholic steatohepatitis. Other paracrine messengers from neighboring mesenchymal cells include HGF, FGF, and TGFα and β. Interestingly, these factors appear to have opposite effects on hepatocytes and progenitors, which may explain the regulatory mechanisms that transfer regeneration from one compartment to the other. Extracellular matrix arrives from surrounding cells is also thought to be important. Nevertheless, while there have been a wealth of studies on the mechanisms that regulate activation, proliferation, migration and differentiation of progenitor cells, translation into clinical intervention has not been forthcoming, underlying the complexities of manipulating network regulation.

Conclusion:
The pursuit of understanding liver regeneration has yielded great progress over the last few decades. Technology has allowed us to decipher regulatory networks that control regenerative mechanisms, and has opened up options for therapeutic manipulation. This work has tremendous implications for clinical applications in acute liver failure, small for size transplantation, extensive liver resection, and delay of morbidity and mortality for cirrhotic patients. Regardless of whether this can be achieved by transplantation of progenitor cells to regenerate the liver, or supportive cells to enhance native regeneration, or by drugs to augment hepatocyte regeneration, a clear understanding of these mechanisms is needed to avoid tragic clinical complications that may set the field back. In tandem with other diseases, the world is poised to leap into human studies with stem cell therapies, representing the amalgamation of knowledge, hopes and public expectation. The drive to understand liver regeneration so as to be able to make a difference to our patients has never been more intense.

Abbreviations: PH | partial hepatectomy | | | TNF | tumor necrosis factor | | | NF-κB | nuclear factor-kappaB | | | IL6 | interleukin 6 | | | STAT 3 | signal transducer and activator of transcription 3 | | | IKK2 | inhibitor of kappaB kinase 2 | | | EGF | epidermal growth factor | | | TGFα | transforming growth factor alpha | | | HB-EGF | heparin binding EGF-like growth factor | | | HGF | hepatocyte growth factor | | | PI3K | phosphoinositide 3 kinase | | | Pdk1 | phosphoinositide-dependent protein kinase 1 | | | SOCS | suppressor of cytokine signaling | | | SDF | stromal cell-derived factor | | | TWEAK | tumor necrosis factor-like weak inducer of apoptosis |

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