Considerable progress has been made in developing antifibrotic agents and other strategies to treat liver fibrosis; however, significant long-term restoration of functional liver mass has not yet been achieved. Therefore, we investigated whether transplanted hepatic stem/progenitor cells can effectively repopulate the liver with advanced fibrosis/cirrhosis. Stem/progenitor cells derived from fetal livers or mature hepatocytes from DPPIV+ F344 rats were transplanted into DPPIV− rats with thioacetamide (TAA)-induced fibrosis/cirrhosis; rats were sacrificed 1, 2, or 4 months later. Liver tissues were analyzed by histochemistry, hydroxyproline determination, reverse-transcription polymerase chain reaction (RT-PCR), and immunohistochemistry. After chronic TAA administration, DPPIV− F344 rats exhibited progressive fibrosis, cirrhosis, and severe hepatocyte damage. Besides stellate cell activation, increased numbers of stem/progenitor cells (Dlk-1+, AFP+, CD133+, Sox-9+, FoxJ1+) were observed. In conjunction with partial hepatectomy (PH), transplanted stem/progenitor cells engrafted, proliferated competitively compared to host hepatocytes, differentiated into hepatocytic and biliary epithelial cells, and generated new liver mass with extensive long-term liver repopulation (40.8 ± 10.3%). Remarkably, more than 20% liver repopulation was achieved in the absence of PH, associated with reduced fibrogenic activity (e.g., expression of alpha smooth muscle actin, platelet-derived growth factor receptor β, desmin, vimentin, tissue inhibitor of metalloproteinase-1) and fibrosis (reduced collagen). Furthermore, hepatocytes can also replace liver mass with advanced fibrosis/cirrhosis, but to a lesser extent than fetal liver stem/progenitor cells. Conclusion : This study is a proof of principle demonstration that transplanted epithelial stem/progenitor cells can restore injured parenchyma in a liver environment with advanced fibrosis/cirrhosis and exhibit antifibrotic effects.

Activin A, a multifunctional cytokine, plays an important role in hepatocyte growth suppression and is involved in liver size control. The present study was aimed to determine the cell location of activin A in the normal rat liver microenvironment and the contribution of activin A signaling to the hepatocyte phenotype to obtain insight into molecular mechanisms. Immunohistochemical andin situhybridization analyses identified hepatocytes as the major activin A‐positive cell population in normal liver and identified mast cells as an additional activin A source. To investigate paracrine and autocrine activin A‐stimulated effects, hepatocytes were cocultured with engineered activin A‐secreting cell lines (RF1, TL8) or transduced with an adeno‐associated virus vector encoding activin βA, which led to strikingly altered expression of cell cycle‐related genes (Ki‐67, E2F transcription factor 1 [E2F1], minichromosome maintenance complex component 2 [Mcm2], forkhead box M1 [FoxM1]) and senescence‐related genes (cyclin‐dependent kinase inhibitor 2B [p15^INK4b/CDKN2B], differentiated embryo‐chondrocyte expressed gene 1 [DEC1]) and reduced proliferation and induction of senescence. Microarray analyses identified 453 differentially expressed genes, many of which were not yet recognized as activin A downstream targets (e.g., ADAM metallopeptidase domain 12 [Adam12], semaphorin 7A [Sema7a], LIM and cysteine‐rich domains‐1 [Lmcd1], DAB2, clathrin adaptor protein [Dab2]). Among the main activin A‐mediated molecular/cellular functions are cellular growth/proliferation and movement, molecular transport, and metabolic processes containing highly down‐regulated genes, such as cytochrome P450, subfamily 2, polypeptide 11 (Cyp2C11), sulfotransferase family 1A, member 1 (Sult1a1), glycine‐N‐acyltransferase (Glyat), and bile acid‐CoA:amino acid N‐acyltransferase (Baat). Moreover, Ingenuity Pathway Analyses identified particular gene networks regulated by hepatocyte nuclear factor (HNF)‐4α and peroxisome proliferator‐activated receptor gamma (PPARγ) as key targets of activin A signaling.Conclusion: Ourin vitromodels demonstrated that activin A‐stimulated growth inhibition and cellular senescence is mediated through p15^INK4b/CDKN2Band is associated with up‐ and down‐regulation of numerous target genes involved in multiple biological processes performed by hepatocytes, suggesting that activin A fulfills a critical role in normal liver function. (Hepatology Communications2017;1:852‐870)

The activation of hypoxia-inducible factors (HIF)-1α and 2α in the liver is closely linked to the progression of fatty liver diseases. Prior studies indicated that disrupting hepatocyte HIF-2α attenuates diet-induced hepatic steatosis, subsequently decreasing fibrosis. However, the direct role of hepatocyte HIF-2α in liver fibrosis has not been addressed. Hepatic HIF-2α expression was examined in mouse model of carbon tetrachloride (CCl₄)-induced liver fibrosis. Conditional hepatocyte Hif-2α knockout mice were employed to investigate the role of hepatocyte HIF-2α in fibrosis. Markers of apoptosis, proliferation, inflammation, and fibrosis were assessed through biochemical, molecular, and histological analyses. We found an induction of HIF-2α in CCL₄-injected liver injury and fibrosis mouse models. Hepatocyte-specific deletion of HIF-2α attenuated stellate cell activation and fibrosis, with no significant difference in inflammation. Disrupting hepatocyte HIF-2α led to reduced injury-mediated hepatocellular apoptosis. Surviving hepatocytes exhibited hypertrophy, which was strongly associated with the activation of c-JUN signaling. Our study demonstrates a direct role of hepatocyte HIF-2α in liver fibrosis by promoting hepatocyte apoptosis. The reduction in apoptosis and induction of hepatocyte hypertrophy following HIF-2α disruption is closely linked to enhanced c-JUN signaling, a survival mechanism in response to liver injury. These findings highlight HIF-2α as a potential therapeutic target for liver fibrosis.

Because of their high regenerative potential, stem cells are an ideal resource for development of therapies that replace injured tissue mass and restore function in patients with end‐stage liver diseases. Using a rat model of bile duct ligation (BDL) and biliary fibrosis, we investigated cell engraftment, liver repopulation, and ectopic tissue formation after intrasplenic transplantation of epithelial stem/progenitor cells. Fetal liver cells were infused into the spleens of Fisher 344 rats with progressing biliary fibrosis induced by common BDL or rats without BDL. Cell delivery was well tolerated. After migration to the liver, donor‐derived stem/progenitor cells engrafted, differentiated into hepatocytes and cholangiocytes, and formed large cell clusters at 2 months in BDL rats but not controls. Substantial numbers of donor cells were also detected at the splenic injection site where they generated hepatic and nonhepatic tissue. Transplanted cells differentiated into phenotypes other than hepato/cholangiocytic cells only in rats that underwent BDL. Quantitative reverse‐transcription polymerase chain reaction analyses demonstrated marked up‐regulation of tissue‐specific genes of nonhepatic endodermal lineages (e.g., caudal type homeobox 2 [Cdx2], pancreatic and duodenal homeobox 1 [Pdx1], keratin 13 [CK‐13]), confirmed by immunohistochemistry. Conclusion: BDL and its induced fibrosis promote liver repopulation by ectopically transplanted fetal liver‐derived cells. These cell fractions contain multipotent stem cells that colonize the spleen of BDL rats and differentiate into multiple gastrointestinal tissues, including liver, pancreas, intestine, and esophagus. The splenic microenvironment, therefore, represents an ideal niche to assess the differentiation of these stem cells, while BDL provides a stimulus that induces their differentiation.