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Direct routes to liver-like cells Two groups report new approaches that could lead to the generation of hepatocyte-like cells for liver engineering and regenerative medicine. Lijian Hui and colleagues use a combination of Gata4, Hnf1a and Foxa3 overexpression and p19Arf inactivation to convert mouse fibroblasts directly into induced hepatic (iHep) cells that have gene-expression profiles close to that of mature hepatocytes. Sayaka Sekiya and Atsushi Suzuki identify three combinations of two transcription factors, comprising Hnf4a plus Foxa1, Foxa2 or Foxa3, that can convert mouse embryonic and adult fibroblasts directly into functional iHep cells. Both groups show that when iHep cells are transplanted into mice with a gene deficiency that models liver injury, the cells are able to repopulate the livers and restore their function. The location and timing of cellular differentiation must be stringently controlled for proper organ formation. Normally, hepatocytes differentiate from hepatic progenitor cells to form the liver during development 1 , 2 . However, previous studies have shown that the hepatic program can also be activated in non-hepatic lineage cells after exposure to particular stimuli or fusion with hepatocytes 3 , 4 , 5 , 6 , 7 , 8 , 9 . These unexpected findings suggest that factors critical to hepatocyte differentiation exist and become activated to induce hepatocyte-specific properties in different cell types. Here, by screening the effects of twelve candidate factors, we identify three specific combinations of two transcription factors, comprising Hnf4α plus Foxa1, Foxa2 or Foxa3, that can convert mouse embryonic and adult fibroblasts into cells that closely resemble hepatocytes in vitro . The induced hepatocyte-like (iHep) cells have multiple hepatocyte-specific features and reconstitute damaged hepatic tissues after transplantation. The generation of iHep cells may provide insights into the molecular nature of hepatocyte differentiation and potential therapies for liver diseases.

The liver has a unique ability to regenerate 1 , 2 ; however, in the setting of acute liver failure (ALF), this regenerative capacity is often overwhelmed, leaving emergency liver transplantation as the only curative option 3 – 5 . Here, to advance understanding of human liver regeneration, we use paired single-nucleus RNA sequencing combined with spatial profiling of healthy and ALF explant human livers to generate a single-cell, pan-lineage atlas of human liver regeneration. We uncover a novel ANXA2 + migratory hepatocyte subpopulation, which emerges during human liver regeneration, and a corollary subpopulation in a mouse model of acetaminophen (APAP)-induced liver regeneration. Interrogation of necrotic wound closure and hepatocyte proliferation across multiple timepoints following APAP-induced liver injury in mice demonstrates that wound closure precedes hepatocyte proliferation. Four-dimensional intravital imaging of APAP-induced mouse liver injury identifies motile hepatocytes at the edge of the necrotic area, enabling collective migration of the hepatocyte sheet to effect wound closure. Depletion of hepatocyte ANXA2 reduces hepatocyte growth factor-induced human and mouse hepatocyte migration in vitro, and abrogates necrotic wound closure following APAP-induced mouse liver injury. Together, our work dissects unanticipated aspects of liver regeneration, demonstrating an uncoupling of wound closure and hepatocyte proliferation and uncovering a novel migratory hepatocyte subpopulation that mediates wound closure following liver injury. Therapies designed to promote rapid reconstitution of normal hepatic microarchitecture and reparation of the gut–liver barrier may advance new areas of therapeutic discovery in regenerative medicine. Harnessing single-nucleus RNA sequencing and spatial profiling, this work dissects unanticipated aspects of human liver regeneration to uncover a novel migratory hepatocyte subpopulation mediating wound closure following acute liver injury.

Normal cells become cancer cells after a malignant transformation, but whether cancer cells can be reversed to normal status remains elusive. Here, we report that the combination of hepatocyte nuclear factor 1A (HNF1A), HNF4A and forkhead box protein A3 (FOXA3) synergistically reprograms hepatocellular carcinoma (HCC) cells to hepatocyte-like cells (reprogrammed hepatocytes, rHeps). Our results show that rHeps lose the malignant phenotypes of cancer cells and retrieve hepatocyte-specific characteristics including hepatocyte-like morphology; global expression pattern of genes and specific biomarkers of hepatocytes; and the unique hepatic functions of albumin (ALB) secretion, glycogen synthesis, low-density lipoprotein (LDL) uptake, urea production, cytochrome P450 enzymes induction and drug metabolism. Intratumoral injection of these three factors efficiently shrank patient-derived tumor xenografts and reprogrammed HCC cells in vivo. Most importantly, transplantation of rHeps in the liver of fumarylacetoacetate hydrolase-deficient (Fah−/−) mice led to the reconstruction of hepatic lobules and the restoration of hepatic function. Mechanistically, exogenous expression of HNF1A, HNF4A and FOXA3 in HCC cells initiated the endogenous expression of numerous hepatocyte nuclear factors, which promoted the conversion of HCC cells to hepatocyte-like cells. Collectively, our results indicate the successful conversion of hepatoma cells to hepatocyte-like cells, not only extending our current knowledge of cell reprogramming but also providing a route towards a novel therapeutic strategy for cancer.

The cause of organ failure is enigmatic for many degenerative diseases, including end-stage liver disease. Here, using a CCl4-induced rat model of irreversible and fatal hepatic failure, which also exhibits terminal changes in the extracellular matrix, we demonstrated that chronic injury stably reprograms the critical balance of transcription factors and that diseased and dedifferentiated cells can be returned to normal function by re-expression of critical transcription factors, a process similar to the type of reprogramming that induces somatic cells to become pluripotent or to change their cell lineage. Forced re-expression of the transcription factor HNF4α induced expression of the other hepatocyte-expressed transcription factors; restored functionality in terminally diseased hepatocytes isolated from CCl4-treated rats; and rapidly reversed fatal liver failure in CCl4-treated animals by restoring diseased hepatocytes rather than replacing them with new hepatocytes or stem cells. Together, the results of our study indicate that disruption of the transcription factor network and cellular dedifferentiation likely mediate terminal liver failure and suggest reinstatement of this network has therapeutic potential for correcting organ failure without cell replacement.

HNF4A and HNF1A encode transcription factors that are important for the development and function of the pancreas and liver. Mutations in both genes have been directly linked to Maturity Onset Diabetes of the Young (MODY) and type 2 diabetes (T2D) risk. To better define the pleiotropic gene regulatory roles of HNF4A and HNF1A, we generated a comprehensive genome-wide map of their binding targets in pancreatic and hepatic cells using ChIP-Seq. HNF4A was found to bind and regulate known ( ACY3 , HAAO, HNF1A , MAP3K11 ) and previously unidentified ( ABCD3 , CDKN2AIP , USH1C , VIL1 ) loci in a tissue-dependent manner. Functional follow-up highlighted a potential role for HAAO and USH1C as regulators of beta cell function. Unlike the loss-of-function HNF4A/MODY1 variant I271fs, the T2D-associated HNF4A variant (rs1800961) was found to activate AKAP1 , GAD2 and HOPX gene expression, potentially due to changes in DNA-binding affinity. We also found HNF1A to bind to and regulate GPR39 expression in beta cells. Overall, our studies provide a rich resource for uncovering downstream molecular targets of HNF4A and HNF1A that may contribute to beta cell or hepatic cell (dys)function, and set up a framework for gene discovery and functional validation. Here, the authors generated a genome-wide map of the global targets bound by HNF4A and HNF1A in beta cells and hepatic cells, and highlighted notable downstream pathways and target genes that may influence beta cell function. This approach also shed light on a potentially activating effect of a HNF4A type 2 diabetes risk variant.

Direct routes to liver-like cells Two groups report new approaches that could lead to the generation of hepatocyte-like cells for liver engineering and regenerative medicine. Lijian Hui and colleagues use a combination of Gata4, Hnf1a and Foxa3 overexpression and p19Arf inactivation to convert mouse fibroblasts directly into induced hepatic (iHep) cells that have gene-expression profiles close to that of mature hepatocytes. Sayaka Sekiya and Atsushi Suzuki identify three combinations of two transcription factors, comprising Hnf4a plus Foxa1, Foxa2 or Foxa3, that can convert mouse embryonic and adult fibroblasts directly into functional iHep cells. Both groups show that when iHep cells are transplanted into mice with a gene deficiency that models liver injury, the cells are able to repopulate the livers and restore their function. The generation of functional hepatocytes independent of donor liver organs is of great therapeutic interest with regard to regenerative medicine and possible cures for liver disease 1 . Induced hepatic differentiation has been achieved previously using embryonic stem cells or induced pluripotent stem cells 2 , 3 , 4 , 5 , 6 , 7 , 8 . Particularly, hepatocytes generated from a patient’s own induced pluripotent stem cells could theoretically avoid immunological rejection. However, the induction of hepatocytes from induced pluripotent stem cells is a complicated process that would probably be replaced with the arrival of improved technology. Overexpression of lineage-specific transcription factors directly converts terminally differentiated cells into some other lineages 9 , 10 , 11 , 12 , including neurons 13 , cardiomyocytes 14 and blood progenitors 15 ; however, it remains unclear whether these lineage-converted cells could repair damaged tissues in vivo . Here we demonstrate the direct induction of functional hepatocyte-like (iHep) cells from mouse tail-tip fibroblasts by transduction of Gata4, Hnf1α and Foxa3, and inactivation of p19 Arf . iHep cells show typical epithelial morphology, express hepatic genes and acquire hepatocyte functions. Notably, transplanted iHep cells repopulate the livers of fumarylacetoacetate-hydrolase-deficient ( Fah −/− ) mice and rescue almost half of recipients from death by restoring liver functions. Our study provides a novel strategy to generate functional hepatocyte-like cells for the purpose of liver engineering and regenerative medicine.

The Foxa subfamily of winged helix/forkhead box (Fox) transcription factors has been the subject of genetic and biochemical study for over 15 years. During this time its three members, Foxa1, Foxa2 and Foxa3, have been found to play important roles in multiple stages of mammalian life, beginning with early development, continuing during organogenesis, and finally in metabolism and homeostasis in the adult. Foxa2 is required for the formation of the node and notochord, and in its absence severe defects in gastrulation, neural tube patterning, and gut morphogenesis result in embryonic lethality. Foxa1 and Foxa2 cooperate to establish competence in foregut endoderm and are required for normal development of endoderm-derived organs such as the liver, pancreas, lungs, and prostate. In post-natal life, members of the Foxa family control glucose metabolism through the regulation of multiple target genes in the liver, pancreas, and adipose tissue. Insight into the unique molecular basis of Foxa function has been obtained from recent genetic and genomic data, which identify the Foxa proteins as 'pioneer factors' whose binding to promoters and enhancers enable chromatin access for other tissue-specific transcription factors.

Gain-of-function mutations in isocitrate dehydrogenase ( IDH ) are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly cancer of the liver bile ducts; now mutant IDH is shown to block liver cell differentiation through the suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence, leading to expansion of liver progenitor cells primed for progression to IHCC. Mechanism of induction of a liver cancer Cancer-associated gain-of-function isocitrate dehydrogenase (IDH) mutations produce the 'oncometabolite' 2-hydroxyglutarate (2HG) that can inhibit a-ketoglutarate-dependent dioxygenase enzymes. Nabeel Bardeesy and colleagues show here that 2HG plays an active role in carcinogenesis: mutant IDH blocks liver progenitor cells from undergoing hepatocyte lineage progression through the production of 2HG and suppression of HNF4a, a master regulator of hepatocyte differentiation. Moreover, where mutant IDH coexists with activated Kras , it drives the expansion of liver progenitor cells, development of premalignant biliary lesions and progression to metastatic intrahepatic cholangiocarcinoma. The transgenic mouse model used here should facilitate further study of IDH function, particularly important in relation to cholangiocarcinoma, which is resistant to current treatments. Mutations in isocitrate dehydrogenase 1 ( IDH1 ) and IDH2 are among the most common genetic alterations in intrahepatic cholangiocarcinoma (IHCC), a deadly liver cancer 1 , 2 , 3 , 4 , 5 . Mutant IDH proteins in IHCC and other malignancies acquire an abnormal enzymatic activity allowing them to convert α-ketoglutarate (αKG) to 2-hydroxyglutarate (2HG), which inhibits the activity of multiple αKG-dependent dioxygenases, and results in alterations in cell differentiation, survival, and extracellular matrix maturation 6 , 7 , 8 , 9 , 10 . However, the molecular pathways by which IDH mutations lead to tumour formation remain unclear. Here we show that mutant IDH blocks liver progenitor cells from undergoing hepatocyte differentiation through the production of 2HG and suppression of HNF-4α, a master regulator of hepatocyte identity and quiescence. Correspondingly, genetically engineered mouse models expressing mutant IDH in the adult liver show an aberrant response to hepatic injury, characterized by HNF-4α silencing, impaired hepatocyte differentiation, and markedly elevated levels of cell proliferation. Moreover, IDH and Kras mutations, genetic alterations that co-exist in a subset of human IHCCs 4 , 5 , cooperate to drive the expansion of liver progenitor cells, development of premalignant biliary lesions, and progression to metastatic IHCC. These studies provide a functional link between IDH mutations, hepatic cell fate, and IHCC pathogenesis, and present a novel genetically engineered mouse model of IDH-driven malignancy.

Intracellular iron homeostasis imbalance is linked to cellular ferroptosis and inflammatory injury diseases. NCOA4-mediated ferritin autophagy is vital for regulating intracellular iron homeostasis, but its impact on acute liver failure (ALF) pathogenesis and regulatory mechanisms remain unclear. This study explores the role and regulatory mechanisms of NCOA4 in hepatocyte ferroptosis and ALF progression. To investigate the relationship between NCOA4 expression and acute liver failure (ALF), we compared the protein expression levels in normal and pathological liver tissues. By establishing cell and mouse models, we determined the correlation among NCOA4 expression, ferroptosis, and inflammatory liver injury. Additionally, we explored the regulatory effect of NCOA4 on hepatocyte ferroptosis by interfering with gene expression and observing mitochondrial structure changes. Finally, we evaluated the regulation of NCOA4 expression and its protective effect against acute inflammatory injury in hepatocytes. Our results showed that NCOA4 expression was significantly higher in patients with hepatitis B virus - related acute - on - chronic liver failure (HBV - ACLF) compared to those with chronic hepatitis B. Similarly, NCOA4 was upregulated in ALF model mice and inflammatory hepatocytes. Silencing NCOA4 alleviated LPS - induced ferroptosis in inflammatory hepatocytes. Mechanistic research indicated that the transcription of hepatic nuclear factor 4 A (HNF4A) negatively regulated NCOA4. HNF4A transcriptionally inhibited NCOA4 expression, reducing hepatocyte ferroptosis through anti - ferritin autophagy. This study identified the HNF4A - NCOA4 axis and ferritinophagy as crucial factors in hepatocyte ferroptosis and the pathogenesis of acute liver failure (ALF). These findings suggest that the HNF4A - NCOA4 axis could be a potential therapeutic target for ALF.