Sriram Bandi

Phenotype reversion in fetal human liver epithelial cells identifies the role of an intermediate meso-endodermal stage before hepatic maturation

Understanding the biological potential of fetal stem/progenitor cells will help define mechanisms in liver development and homeostasis. We isolated epithelial fetal human liver cells and established phenotype-specific changes in gene expression during continuous culture conditions. Fetal human liver epithelial cells displayed stem cell properties with multilineage gene expression, extensive proliferation and generation of mesenchymal lineage cells, although the initial epithelial phenotype was rapidly supplanted by meso-endodermal phenotype in culture. This meso-endodermal phenotype was genetically regulated through cytokine signaling, including transforming growth factor β, bone morphogenetic protein, fibroblast growth factor and other signaling pathways. Reactivation of HNF3α (FOXA1) transcription factor, a driver of hepatic specification in the primitive endoderm, indicated that the meso-endodermal phenotype represented an earlier developmental stage of cells. We found that fetal liver epithelial cells formed mature hepatocytes in vivo, including after genetic manipulation using lentiviral vectors, offering convenient assays for analysis of further cell differentiation and fate. Taken together, these studies demonstrate plasticity in fetal liver epithelial stem cells, offer paradigms for defining mechanisms regulating lineage switching in stem cells, and provide potential avenues for regulating cell phenotypes for applications of stem cells, such as for cell therapy.

Perturbations in Ataxia Telangiectasia Mutant Signaling Pathways After Drug-Induced Acute Liver Failure and Their Reversal During Rescue of Animals by Cell Therapy

Superior insights into molecular mechanisms of liver failure, which are not fully understood, will help strategies for inducing liver regeneration. We examined hepatotoxic mechanisms in mice homozygous for the severe combined immune deficiency mutation in the protein kinase, DNA-activated, catalytic polypeptide. Mice were treated with rifampicin, phenytoin, and monocrotaline. The ensuing acute liver failure was characterized by serological, histological, and mRNA studies. Subsequently, we studied whether transplantation of hepatocytes could rescue animals with liver failure. We found extensive liver damage in these animals, with mortality over several days. The expression of multiple hepatic genes was rapidly altered, including those representing pathways in oxidative/metabolic stress, inflammation, DNA damage-repair, and ataxia telangiectasia mutant (Atm) signaling pathways. This led to liver cell growth arrest involving cyclin-dependent kinase inhibitor 1A. Transplantation of hepatocytes with microcarriers in the peritoneal cavity efficiently rescued animals with liver failure. Molecular abnormalities rapidly reversed, including in hepatic Atm and downstream signaling pathways; and residual hepatocytes overcame cyclin-dependent kinase inhibitor 1A-induced cell growth arrest. Reseeding of the liver with transplanted hepatocytes was not required for rescue because native hepatocytes overcame cell growth-arrest to regenerate the liver. This likely resulted from paracrine signaling from hepatocytes in the peritoneal cavity. We concluded that Atm signaling played critical roles in the pathological features of liver failure. These studies should help redirect examination of pathophysiologic and therapeutic mechanisms in liver failure.

Hepatic stellate cells promote hepatocyte engraftment in rat liver after prostaglandin-endoperoxide synthase inhibition

BACKGROUND & AIMS Hepatic inflammation occurs immediately after cells are transplanted to the liver, but the mechanisms that underlie this process are not fully defined. We examined cyclooxygenase pathways that mediate hepatic inflammation through synthesis of prostaglandins, prostacyclins, thromboxanes, and other prostanoids following transplantation of hepatocytes. METHODS We transplanted F344 rat hepatocytes into syngeneic dipeptidyl peptidase IV-deficient F344 rats. Changes in cyclooxygenase pathways were analyzed, and specific pathways were blocked pharmacologically; the effects on cell engraftment and native liver cells were determined. RESULTS Transplantation of hepatocytes induced hepatic expression of prostaglandin-endoperoxide synthases 1 and 2, which catalyze production of prostaglandin H2, as well as the downstream factor thromboxane synthase, which produces thromboxane A2 (a regulator of vascular and platelet responses in inflammation). Transplanted hepatocytes were in proximity with liver cells that expressed prostaglandin-endoperoxide synthases. The number of engrafted hepatocytes increased in rats given naproxen or celecoxib before transplantation but not in rats given furegrelate (an inhibitor of thromboxane synthase) or clopodigrel (an antiplatelet drug). Naproxen and celecoxib did not prevent hepatic ischemia or activation of neutrophils, Kupffer cells, or inflammatory cytokines, but they did induce hepatic stellate cells to express cytoprotective genes, vascular endothelial growth factor and hepatocyte growth factor, and matrix-type metalloproteinases and tissue inhibitor of metalloproteinase-1, which regulate hepatic remodeling. CONCLUSIONS Activation of cyclooxygenase pathways interferes with engraftment of transplanted hepatocytes in the liver. Pharmacologic blockade of prostaglandin-endoperoxide synthases stimulated hepatic stellate cells and improved cell engraftment.

Directly acting drugs prostacyclin or nitroglycerine and endothelin receptor blocker bosentan improve cell engraftment in rodent liver

To optimize strategies for liver‐directed cell therapy, prevention of initial transplanted cell losses is particularly important for subsequent liver repopulation. After cell transplantation in hepatic sinusoids, perturbations in hepatic microcirculation along with changes in various liver cell types are among the earliest changes. Therefore, for advancing further concepts in cell engraftment we studied vascular and related events in the liver after transplanting syngeneic hepatocytes into dipeptidyl peptidase IV‐deficient rats. We treated rats with vascular drugs to define whether deleterious cell transplantation‐induced events could be controlled followed by improvements in transplanted cell engraftment and proliferation. We found cell transplantation altered liver gene expression related to vessel tone, inflammation, cell adhesion, thrombosis, or tissue damage/remodeling. This was due to hepatic ischemia, endothelial injury, and activation of neutrophils, Kupffer cells, and hepatic stellate cells. Treatment of rats before cell transplantation with the angiotensin converting enzyme blocker, lisinopril, or angiotensin II receptor blocker, losartan, did not improve cell engraftment. By contrast, direct‐acting nitroglycerine or prostacyclin improved cell engraftment and also kinetics of liver repopulation. These drugs lowered hepatic ischemia and inflammation, whereas pretreatment of rats with the dual endothelin‐1 receptor blocker, bosentan, improved cell engraftment independently of hepatic ischemia or inflammation, without improving liver repopulation. However, incubation of hepatocytes with bosentan protected cells from cytokine toxicity in vitro and produced superior cell engraftment and proliferation in vivo. Conclusion: Cell transplantation‐induced changes in hepatic microcirculation contributed to transplanted cell clearances from liver. Vascular drugs, such as nitroglycerine, prostacyclin, and bosentan, offer opportunities for improving cell therapy results through superior cell engraftment and liver repopulation. Ongoing clinical use of these drugs will permit rapid translation of the findings in people. (HEPATOLOGY 2013)

Spontaneous origin from human embryonic stem cells of liver cells displaying conjoint meso-endodermal phenotype with hepatic functions.

Understanding the identity of lineage-specific cells arising during manipulations of stem cells is necessary for developing their potential applications. For instance, replacement of crucial functions in organ failure by transplantation of suitable stem-cell-derived cells will be applicable to numerous disorders, but requires insights into the origin, function and fate of specific cell populations. We studied mechanisms by which the identity of differentiated cells arising from stem cells could be verified in the context of natural liver-specific stem cells and whether such differentiated cells could be effective for supporting the liver following cell therapy in a mouse model of drug-induced acute liver failure. By comparing the identity of naturally occurring fetal human liver stem cells, we found that cells arising in cultures of human embryonic stem cells (hESCs) recapitulated an early fetal stage of liver cells, which was characterized by conjoint meso-endoderm properties. Despite this fetal stage, hESC-derived cells could provide liver support with appropriate metabolic and ammonia-fixation functions, as well as cytoprotection, such that mice were rescued from acute liver failure. Therefore, spontaneous or induced differentiation of human embryonic stem cells along the hepatic endoderm will require transition through fetal-like stages. This offers opportunities to prospectively identify whether suitable cells have been generated through manipulation of stem cells for cell therapy and other applications.

Arsenic trioxide amplifies cisplatin toxicity in human tubular cells transformed by HPV-16 E6/E7 for further therapeutic directions in renal cell carcinoma

Human papillomavirus (HPV) DNA integrations may affect therapeutic responses in cancers through ATM network-related DNA damage response (DDR). We studied whether cisplatin-induced DDR was altered in human HK-2 renal tubular cells immortalized by HPV16 E6/E7 genes. Cytotoxicity assays utilized thiazolyl blue dye and DDR was identified by gene expression differences, double-strand DNA breaks, ATM promoter activity, and analysis of cell cycling and side population cells. After cisplatin, HK-2 cells showed greater ATM promoter activity indicating activation of this network, but DDR was muted, since little γH2AX was expressed, DNA strand breaks were absent and cells continued cycling. When HK-2 cells were treated with the MDM2 antagonist inducing p53, nutlin-3, or p53 transcriptional activator, tenovin-1, cell growth decreased but cisplatin toxicity was unaffected. By contrast, arsenic trioxide, which by inhibiting wild-type p53-induced phosphatase-1 that serves responses downstream of p53, and by depolymerizing tubulin, synergistically enhanced cisplatin cytotoxicity including loss of SP cells. Our findings demonstrated that HPV16 E6/E7 altered DDR through p53-mediated cell growth controls, which may be overcome by targeting of WIP1 and other processes, and thus should be relevant for treating renal cell carcinoma.

Endogenous antiviral microRNAs determine permissiveness for hepatitis B virus replication in cultured human fetal and adult hepatocytes

Superior cell culture models for hepatitis B virus (HBV) will help advance insights into host‐virus interactions. To identify mechanisms regulating HBV replication, this study used cultured human HepG2 cells and adult or fetal hepatocytes transduced with adenoviral vector to express HBV upstream of green fluorescent protein. The vector efficiently transduced all cell types. In HepG2 cells, replicative viral intermediates, nucleocapsid‐associated HBcAg, and HBsAg were expressed. However, in fetal or adult hepatocytes, pregenomic HBV RNA and viral RNAs were expressed, but nucleocapsid‐associated HBcAg in cells or HBsAg in culture medium were absent, indicating interruptions in viral replication due to possible microRNA‐related interference. MicroRNA profiling demonstrated that a large number of microRNAs with antiviral potential were differentially expressed in hepatocytes after culture. In transfection assays using HepG2 cells, candidate antiviral microRNAs, e.g., hsa‐miR‐24 or hsa‐miR‐638 decreased the levels of HBV transcripts or HBV gene products. Since candidate microRNAs could have targeted interferon response genes as an alternative explanation interferon signaling was examined. However, HBV replication in cultured hepatocytes was not restored despite successful inhibition of JAK1/2‐STAT signaling by the inhibitor, ruxolitinib. Therefore, HBV was unable to complete replication in cultured hepatocytes due to expression of multiple antiviral microRNAs. This mechanism should help understand restrictions in HBV replication for developing HBV models in cultured cells while providing frameworks for pathophysiological studies of HBV replication in subsets of hepatocytes or stem/progenitor cells during hepatitis. J. Med. Virol. 87:1168–1183, 2015.

Evaluation of Cytotoxicity and DNA Damage Response with Analysis of Intracellular ATM Signaling Pathways

Maintenance of genome integrity by preventing and overcoming DNA damage is critical for cell survival. Deficiency or aberrancy in the DNA damage response, for example, through ataxia telangiectasia mutated (ATM) signaling, lead to pathophysiological perturbations in organs throughout the body. Therefore, control of DNA damage is of major interest for development of therapeutic agents. Such efforts will greatly benefit from convenient and simple diagnostic and/or drug development tools to demonstrate whether ATM and related genes have been activated and to then determine whether these have been returned to normal levels of activity because pathway members sense and also repair DNA damage. To overcome difficulties in analyzing differences in multitudinous ATM pathway members following DNA damage, we measured ATM promoter activity with a fluorescent td-Tomato reporter gene to interrogate the global effects of ATM signaling pathways. In cultured HuH-7 cell line derived from human hepatocellular carcinoma, cis-platinum, acetaminophen, or hydrogen peroxide caused DNA strand breaks and ATM pathway activation as shown by γH2AX expression, which in turn, led to rapid and sustained increases in ATM promoter activity. This assay of ATM promoter activity identified biological agents capable of controlling cellular DNA damage in toxin-treated HuH-7 cells and in mice after onset of drug-induced acute liver failure. Therefore, the proposed assay of ATM promoter activity in HuH-7 cells was appropriately informative for treating DNA damage. High-throughput screens using ATM promoter activation will be helpful for therapeutic development in DNA damage-associated abnormal ATM signaling in various cell types and organs.

Ischemic preconditioning affects long-term cell fate through DNA damage-related molecular signaling and altered proliferation.

Despite the potential of ischemic preconditioning for organ protection, long-term effects in terms of molecular processes and cell fates are ill defined. We determined consequences of hepatic ischemic preconditioning in rats, including cell transplantation assays. Ischemic preconditioning induced persistent alterations; for example, after 5 days liver histology was normal, but γ-glutamyl transpeptidase expression was observed, with altered antioxidant enzyme content, lipid peroxidation, and oxidative DNA adducts. Nonetheless, ischemic preconditioning partially protected from toxic liver injury. Similarly, primary hepatocytes from donor livers preconditioned with ischemia exhibited undesirably altered antioxidant enzyme content and lipid peroxidation, but better withstood insults. However, donor hepatocytes from livers preconditioned with ischemia did not engraft better than hepatocytes from control livers. Moreover, proliferation of hepatocytes from donor livers preconditioned with ischemia decreased under liver repopulation conditions. Hepatocytes from donor livers preconditioned with ischemia showed oxidative DNA damage with expression of genes involved in MAPK signaling that impose G1/S and G2/M checkpoint restrictions, including p38 MAPK-regulated or ERK-1/2-regulated cell-cycle genes such as FOS, MAPK8, MYC, various cyclins, CDKN2A, CDKN2B, TP53, and RB1. Thus, although ischemic preconditioning allowed hepatocytes to better withstand secondary insults, accompanying DNA damage and molecular events simultaneously impaired their proliferation capacity over the long term. Mitigation of ischemic preconditioning-induced DNA damage and deleterious molecular perturbations holds promise for advancing clinical applications.

Molecular perturbations restrict potential for liver repopulation of hepatocytes isolated from non-heart-beating donor rats

Organs from non–heart‐beating donors are attractive for use in cell therapy. Understanding the nature of molecular perturbations following reperfusion/reoxygenation will be highly significant for non–heart‐beating donor cells. We studied non–heart‐beating donor rats for global gene expression with Affymetrix microarrays, hepatic tissue integrity, viability of isolated hepatocytes, and engraftment and proliferation of transplanted cells in dipeptidyl peptidase IV‐deficient rats. In non–heart‐beating donors, liver tissue was morphologically intact for >24 hours with differential expression of 1, 95, or 372 genes, 4, 16, or 34 hours after death, respectively, compared with heart‐beating donors. These differentially expressed genes constituted prominent groupings in ontological pathways of oxidative phosphorylation, adherence junctions, glycolysis/gluconeogenesis, and other discrete pathways. We successfully isolated viable hepatocytes from non–heart‐beating donors, especially up to 4 hours after death, although the hepatocyte yield and viability were inferior to those of hepatocytes from heart‐beating donors (P < 0.05). Similarly, although hepatocytes from non–heart‐beating donors engrafted and proliferated after transplantation in recipient animals, this was inferior to hepatocytes from heart‐beating donors (P < 0.05). Gene expression profiling in hepatocytes isolated from non–heart‐beating donors showed far greater perturbations compared with corresponding liver tissue, including representation of pathways in focal adhesion, actin cytoskeleton, extracellular matrix–receptor interactions, multiple ligand–receptor interactions, and signaling in insulin, calcium, wnt, Jak‐Stat, or other cascades. Conclusion: Liver tissue remained intact over prolonged periods after death in non–heart‐beating donors, but extensive molecular perturbations following reperfusion/reoxygenation impaired the viability of isolated hepatocytes from these donors. Insights into molecular changes in hepatocytes from non–heart‐beating donors offer opportunities for improving donor cell viability, which will advance the utility of non–heart‐beating donor organs for cell therapy or other applications. (HEPATOLOGY 2012)