Kilangsungla Yanger

Engraftment and Reconstitution of Hematopoiesis Is Dependent on VEGFR2-Mediated Regeneration of Sinusoidal Endothelial Cells

Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.

Hippo Pathway Activity Influences Liver Cell Fate

The Hippo-signaling pathway is an important regulator of cellular proliferation and organ size. However, little is known about the role of this cascade in the control of cell fate. Employing a combination of lineage tracing, clonal analysis, and organoid culture approaches, we demonstrate that Hippo pathway activity is essential for the maintenance of the differentiated hepatocyte state. Remarkably, acute inactivation of Hippo pathway signaling in vivo is sufficient to dedifferentiate, at very high efficiencies, adult hepatocytes into cells bearing progenitor characteristics. These hepatocyte-derived progenitor cells demonstrate self-renewal and engraftment capacity at the single-cell level. We also identify the NOTCH-signaling pathway as a functional important effector downstream of the Hippo transducer YAP. Our findings uncover a potent role for Hippo/YAP signaling in controlling liver cell fate and reveal an unprecedented level of phenotypic plasticity in mature hepatocytes, which has implications for the understanding and manipulation of liver regeneration.

Robust cellular reprogramming occurs spontaneously during liver regeneration

Cellular reprogramming-the ability to interconvert distinct cell types with defined factors-is transforming the field of regenerative medicine. However, this phenomenon has rarely been observed in vivo without exogenous factors. Here, we report that activation of Notch, a signaling pathway that mediates lineage segregation during liver development, is sufficient to reprogram hepatocytes into biliary epithelial cells (BECs). Moreover, using lineage tracing, we show that hepatocytes undergo widespread hepatocyte-to-BEC reprogramming following injuries that provoke a biliary response, a process requiring Notch. These results provide direct evidence that mammalian regeneration prompts extensive and dramatic changes in cellular identity under injury conditions.

Notch Signaling Is Activated in Human Hepatocellular Carcinoma and Induces Tumor Formation in Mice

BACKGROUND & AIMS The Notch signaling pathway is activated in leukemia and solid tumors (such as lung cancer), but little is known about its role in liver cancer. METHODS The intracellular domain of Notch was conditionally expressed in hepatoblasts and their progeny (hepatocytes and cholangiocytes) in mice. This was achieved through Cre expression under the control of an albumin and α-fetoprotein (AFP) enhancer and promoter (AFP-Notch intracellular domain [NICD]). We used comparative functional genomics to integrate transcriptome data from AFP-NICD mice and human hepatocellular carcinoma (HCC) samples (n = 683). A Notch gene signature was generated using the nearest template prediction method. RESULTS AFP-NICD mice developed HCC with 100% penetrance when they were 12 months old. Activation of Notch signaling correlated with activation of 3 promoters of insulin-like growth factor 2; these processes appeared to contribute to hepatocarcinogenesis. Comparative functional genomic analysis identified a signature of Notch activation in 30% of HCC samples from patients. These samples had altered expression in Notch pathway genes and activation of insulin-like growth factor signaling, despite a low frequency of mutations in regions of NOTCH1 associated with cancer. Blocking Notch signaling in liver cancer cells with the Notch activation signature using γ-secretase inhibitors or by expressing a dominant negative form of mastermind-like 1 reduced their proliferation in vitro. CONCLUSIONS Notch signaling is activated in human HCC samples and promotes formation of liver tumors in mice. The Notch signature is a biomarker of response to Notch inhibition in vitro.

Lineage tracing demonstrates no evidence of cholangiocyte epithelial‐to‐mesenchymal transition in murine models of hepatic fibrosis

Whether or not cholangiocytes or their hepatic progenitors undergo an epithelial‐to‐mesenchymal transition (EMT) to become matrix‐producing myofibroblasts during biliary fibrosis is a significant ongoing controversy. To assess whether EMT is active during biliary fibrosis, we used Alfp‐Cre × Rosa26‐YFP mice, in which the epithelial cells of the liver (hepatocytes, cholangiocytes, and their bipotential progenitors) are heritably labeled at high efficiency with yellow fluorescent protein (YFP). Primary cholangiocytes isolated from our reporter strain were able to undergo EMT in vitro when treated with transforming growth factor‐β1 alone or in combination with tumor necrosis factor‐α, as indicated by adoption of fibroblastoid morphology, intracellular relocalization of E‐cadherin, and expression of α‐smooth muscle actin (α‐SMA). To determine whether EMT occurs in vivo, we induced liver fibrosis in Alfp‐Cre × Rosa26‐YFP mice using the bile duct ligation (BDL) (2, 4, and 8 weeks), carbon tetrachloride (CCl4) (3 weeks), and 3,5‐diethoxycarbonyl‐1,4‐dihydrocollidine (DDC; 2 and 3 weeks) models. In no case did we find evidence of colocalization of YFP with the mesenchymal markers S100A4, vimentin, α‐SMA, or procollagen 1α2, although these proteins were abundant in the peribiliary regions. Conclusion: Hepatocytes and cholangiocytes do not undergo EMT in murine models of hepatic fibrosis. (Hepatology 2011;)

Facultative stem cells in liver and pancreas: fact and fancy.

Tissue turnover is a regular feature of higher eukaryotes, either as part of normal wear and tear (homeostasis) or in response to injury (regeneration). Cell replacement is achieved either through replication of existing cells or differentiation from a self‐renewing pool of stem cells. The major distinction regards cellular potential, because stem cells by definition have a capacity to differentiate, while replication implies that cells adopt a single fate under physiologic conditions. A hybrid model, the facultative stem cell (FSC) model, posits that tissues contain cells that normally exhibit unipotency but have the capacity to function as stem cells upon injury. The FSC paradigm is well established in urodele amphibians, but the nature and role of FSCs in mammals is less defined. Here, we review the evidence for FSCs in two mammalian organs, the liver and the pancreas, and discuss alternative models that could account for regeneration in these organs. Developmental Dynamics 240:521–529, 2011.

Cytokinesis defines a spatial landmark for hepatocyte polarization and apical lumen formation.

ABSTRACT By definition, all epithelial cells have apical–basal polarity, but it is unclear how epithelial polarity is acquired and how polarized cells engage in tube formation. Here, we show that hepatocyte polarization is linked to cytokinesis using the rat hepatocyte cell line Can 10. Before abscission, polarity markers are delivered to the site of cell division in a strict spatiotemporal order. Immediately after abscission, daughter cells remain attached through a unique disc-shaped structure, which becomes the site for targeted exocytosis, resulting in the formation of a primitive bile canaliculus. Subsequently, oriented cell division and asymmetric cytokinesis occur at the bile canaliculus midpoint, resulting in its equal partitioning into daughter cells. Finally, successive cycles of oriented cell division and asymmetric cytokinesis lead to the formation of a tubular bile canaliculus, which is shared by two rows of hepatocytes. These findings define a novel mechanism for cytokinesis-linked polarization and tube formation, which appears to be broadly conserved in diverse cell types.

Large tumor suppressor homologs 1 and 2 regulate mouse liver progenitor cell proliferation and maturation through antagonism of the coactivators YAP and TAZ.

In the adult liver, the Hippo pathway mammalian STE20‐like protein kinases 1 and 2 and large tumor suppressor homologs 1 and 2 (LATS1/2) control activation of the transcriptional coactivators Yes‐associated protein (YAP) and WW domain containing transcription regulator 1 (TAZ) in hepatocytes and biliary epithelial cells, thereby regulating liver cell proliferation, differentiation, and malignant transformation. Less is known about the contribution of Hippo signaling to liver development. We used conditional mutagenesis to show that the Hippo signaling pathway kinases LATS1 and LATS2 are redundantly required during mouse liver development to repress YAP and TAZ in both the biliary epithelial and hepatocyte lineages. In the absence of LATS1/2, biliary epithelial cells exhibit excess proliferation while hepatoblasts fail to mature into hepatocytes, defects that result in perinatal lethality. Using an in vitro hepatocyte differentiation assay, we demonstrate that YAP activity decreases and Hippo pathway kinase activities increase upon differentiation. In addition, we show that YAP activation in vitro, resulting from either depletion of its negative regulators LATS1/2 or expression of a mutant form of YAP that is less efficiently phosphorylated by LATS1/2, results in transcriptional suppression of genes that normally accompany hepatocyte maturation. Moreover, we provide evidence that YAP activity is repressed by Hippo pathway activation upon hepatocytic maturation in vitro. Finally, we examine the localization of YAP during fetal liver development and show that higher levels of YAP are found in biliary epithelial cells, while in hepatocytes YAP levels decrease with hepatocyte maturation. Conclusion: Hippo signaling, mediated by the LATS1 and LATS2 kinases, is required to restrict YAP and TAZ activation during both biliary and hepatocyte differentiation. These findings suggest that dynamic regulation of the Hippo signaling pathway plays an important role in differentiation and functional maturation of the liver. (Hepatology 2016;64:1757‐1772).

Abstract 2410: Characterization of Notch as an oncogenic driver in liver

INTRODUCTION: Notch is an evolutionary conserved pathway essential for embryonic development and tissue renewal in metazoan. It plays a key role in liver development by regulating hepatic progenitor cell differentiation. Dysregulation of Notch pathway is found in several cancers, while there are few data regarding its role in hepatocarcinogenesis. AIMS: (1) To study Notch dysregulation in human hepatocellular carcinoma (HCC), and (2) to determine the oncogenic capacity of constitutive Notch activation in transgenic mice. METHODS: Eighteen key Notch pathway genes were evaluated in human samples covering the whole spectrum of human hepatocarcinogenesis (normal liver, cirrhosis, dysplasia and HCC) for DNA copy number changes (Affymetrix 238K, 132 samples) and mRNA levels (Affymetrix U133, 132 samples – Illumina DASL, 164 samples). Notch1 mutation analysis was performed in 50 human HCC samples using Sanger sequencing (GENEWIZ, Inc.). A knock in mouse line carrying the mouse Notch1 intracellular domain (NICD) inserted in the Rosa26 locus downstream of a floxed Neo/STOP codon was crossed with an AFP/albumin-Cre transgenic mouse line. To generate a Notch activation gene signature, we compared the transcriptome of control and transgenic liver samples. The presence of the Notch activation signature was evaluated in human HCC (225 samples) and intrahepatic cholangiocarcinoma (ICC) (149 samples) using the nearest template prediction method. CK19 staining and histopathological features were evaluated by a pathologist in paraffin embedded liver samples from control and transgenic mice. RESULTS: Several Notch genes were significantly up-regulated (corrected p-value 10 months (11/11). Predominant tumor type was HCC, although 30% showed a mixed pattern with progenitor-like cells and CK19 positive staining (3/10). CONCLUSIONS: Notch pathway is significantly dysregulated in human HCC. Genetically engineered mice with constitutive activation of Notch1 undergo liver oncogenesis, suggesting that Notch behaves as an oncogene in liver cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2410. doi:10.1158/1538-7445.AM2011-2410