Studies of liver regeneration following acute liver injury in humans have been limited and mostly focused on events that occur after partial hepatectomy, when mature hepatocyte proliferation is the primary driver of liver repopulation 
. The availability of multiple specimens from the explanted livers of 4 patients who developed HBV-associated ALF gave us a unique opportunity to study the role and extent of HSPC proliferation in liver regeneration associated with this dramatic clinical syndrome. By combining gene expression profiling with liver pathology and clinical data, we provide a molecular definition of liver regeneration following HBV-associated ALF in humans. Although the number of patients included in this study was limited and only a single time point was available for study (at the time of OLT when the native liver was explanted), all patients included had ALF due to the same etiology, all underwent OLT within one week of the clinical onset, and for each of them we analyzed up to 5 liver specimens by microarray and liver histology. Remarkably, we found that different liver specimens from the same patient clustered together when analyzed by MDS and clustering analysis, suggesting the lack of important individual variances among different liver specimens obtained from the same patient.
Gene expression analysis documented a distinct signature associated with ALF. Strikingly, however, when we analyzed all 11,570 genes that passed the filtering criteria by using an unsupervised clustering analysis and MDS, we identified two well-defined clusters that segregated according to the histopathological severity, i.e., MHN vs. SHN. It is important to emphasize that the majority of differentially expressed genes were shared by the two clusters, but they varied in their intensity of expression following a gradient from MHN to SHN to liver donors. One of the most prominent features of the gene signature of HBV-associated ALF was the activation of HSPC, reflecting the extreme conditions under which liver regeneration must occur following massive destruction of the hepatic parenchyma. Interestingly, in agreement with the separation observed between MHN and SHN by MDS and clustering analysis, genes associated with HSPC, most notably CK19, CK7, EPCAM, CD133/PROM1, CD24, and SOX9, showed the highest expression in MHN, whereas the patient with the lowest degree of liver necrosis had the least up-regulation. These findings were corroborated by histopathology, which documented a remarkable ductular reaction in MHN without evidence of hepatocyte-like cells. Thus, our data demonstrate that the strong ductular reaction present in MHN was not associated with further differentiation toward hepatocyte-like cells, a finding also supported by the lack of alpha-fetoprotein gene expression in the liver, as well as by the lack of alpha-fetoprotein increase in serum. In contrast, in SHN besides a prominent ductular reaction, we observed the presence of early regenerative nodules with a hepatocyte-like morphology, reflecting an intermediate phenotype between progenitor cells and hepatocytes. Moreover, the association of intermediate hepatocyte-like cells with elevated serum alpha-fetoprotein levels in Patient 32 reflects the importance of this marker in liver regeneration, as previously reported 
. Recent studies have reported that, in patients with acute and chronic liver disease 
and in mouse models 
, the Wnt and Notch signaling pathways are important in the regulation of HSPC being implicated in the lineage specification of hepatocytes and cholangiocytes, respectively, during the process of liver regeneration 
. In ALF, we found up-regulated genes associated with the Wnt 
and Notch 
pathways, although key components of these pathways were not detected.
Another major finding of our study was a strong gene signature of hepatic fibrogenesis in ALF. Interestingly, genes associated with HSC activation and extracellular matrix components were again more up-regulated in MHN than in SHN, indicating that the extent of hepatic fibrosis positively correlated with the histopathological severity. Consistent with our gene expression profiles, recent data by immunohistochemistry have documented HSC activation in patients with ALF, along with an increase in serum levels of matrix metalloproteinase (MMP-1), MMP-2, MMP-9, and tissue inhibitors of MMP-1 (TIMP-1), TIMP-2 
. Strikingly, MMP-7, whose expression has been found up-regulated in idiopathic pulmonary fibrosis 
as well as in biliary atresia-associated liver fibrosis 
, was the most up-regulated gene in MHN. However, evidence is accumulating to suggest that MMP-7, like other MMPs, has also a major role in cancer 
, although it has also found to be expressed by oval cells in rats models 
. It has been recently hypothesized that the occurrence of fibrosis in ALF may be a physiological and possibly beneficial response of the liver to support the collapsing parenchymal structure through the deposition of new extracellular matrix 
. Our study confirms and further extends this concept providing evidence that the degree of liver fibrosis positively correlates with the histopathological severity. Indeed, the highest expression of genes related to HSC activation and extracellular matrix was detected in patients with MHN.
Cancer has been described as a wound that does not heal 
. Previous studies have investigated similarities and differences between tissue regeneration (wound healing) and tumorigenesis 
. A remarkable feature that we documented in ALF was an overriding tumorigenesis gene signature. Functional analysis using IPA identified more than 900 differentially expressed genes related to cell growth, proliferation and tumorigenesis, most of which were never previously associated with liver regeneration. Interestingly, our analysis also provided evidence that the most-up-regulated genes in MHN reflect a stem/progenitor cell signature, which shares phenotypic traits with cancer stem cells 
, whereas the most up-regulated genes in SHN likely reflect proliferation of more committed hepatocyte-like cells as those present in the early regenerative nodules. One of the most up-regulated genes in ALF, more prominently expressed in SHN, was AKR1B10, a detoxifying enzyme associated with the control of cell growth and proliferation 
. Although the molecular mechanisms underlying its role in cell proliferation and cancer have yet to be elucidated, this gene was found overexpressed in several tumors 
including hepatocellular carcinoma 
. The identification of common genes between liver regeneration and liver cancer underlines the overlaps in the biology and regulation of these processes, emphasizing the need to further dissect the role of these genes as well as the role of stem and progenitor cells in both of these settings 
In summary, gene expression profiling provided new insights into the molecular pathogenesis of liver regeneration in humans. Our study demonstrated that liver regeneration in ALF is characterized by a prominent ductular reaction both in MHN and in SHN, associated with extensive fibrogenesis to repair damaged liver tissue. However, the development of grade IV coma within a few days from the onset of clinical symptoms required an immediate OLT, which prevented the evaluation of whether the regenerative response would have resulted in an effective liver regeneration. Whereas in MHN we have observed a marked HSPC gene signature with no evidence of further differentiation, in SHN the HSPC signature was associated with evidence of more committed hepatocyte-like cells and evidence of highly expressed genes associated with cell growth and proliferation, as well as tumorigenesis, most of them never reported in liver regeneration. Our data provided evidence that the intensity of the gene signature in HBV-associated ALF is directly correlated with the histopathological severity, raising the question whether molecular analysis of tissue samples obtained from patients with ALF prior to transplant could provide prognostic information. Finally, the results of our study underline the importance of dissecting the relationship between liver regeneration and liver cancer.