Abnormal lipid processing but normal long-term repopulation potential of myc-/- hepatocytes.
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ABSTRACT:
SUBMITTER: Edmunds LR
PROVIDER: S-EPMC5058687 | biostudies-literature | 2016 May
REPOSITORIES: biostudies-literature
Publications
Abnormal lipid processing but normal long-term repopulation potential of myc-/- hepatocytes.
Edmunds Lia R LR Otero P Anthony PA Sharma Lokendra L D'Souza Sonia S Dolezal James M JM David Sherin S Lu Jie J Lamm Lauren L Basantani Mahesh M Zhang Pili P Sipula Ian J IJ Li Lucy L Zeng Xuemei X Ding Ying Y Ding Fei F Beck Megan E ME Vockley Jerry J Monga Satdarshan P S SP Kershaw Erin E EE O'Doherty Robert M RM Kratz Lisa E LE Yates Nathan A NA Goetzman Eric P EP Scott Donald D Duncan Andrew W AW Prochownik Edward V EV
Oncotarget 20160501 21
Establishing c-Myc's (Myc) role in liver regeneration has proven difficult particularly since the traditional model of partial hepatectomy may provoke an insufficiently demanding proliferative stress. We used a model of hereditary tyrosinemia whereby the affected parenchyma can be gradually replaced by transplanted hepatocytes, which replicate 50-100-fold, over several months. Prior to transplantation, livers from myc-/- (KO) mice were smaller in young animals and larger in older animals relativ ...[more]
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Project description:Liver transplantation is the only therapeutic option for patients with end-stage liver disease. The shortage of donor organs has led to the search for alternative therapies to restore liver function and bridge patients to transplantation. Our previous work has shown that the proliferation of late gestation E19 fetal hepatocytes is mitogen-independent. This is manifested as differences in the control of ribosome biogenesis, global translation, cell cycle progression and gene expression. In the present study, we investigated whether E19 fetal hepatocytes would engraft and repopulate an injured adult liver. Methods: Fetal hepatocytes were isolated using a monoclonal antibody against a hepatic surface protein, leucine amino peptidase (LAP). LAP+ and LAP- fractions were analyzed by immunofluorescence and microarray. Immunopurified E19 liver cells from DPPIV+ F344 rats were transplanted via splenic injection into partial hepatectomized DPPIV- rats that had been pretreated with mitomycin C. Results: Phenotypic characterization of the LAP+ fetal hepatocytes revealed that more than a third of the isolated cells expressed ductal markers. Transcriptomic analysis revealed that these dual expressing cells represent a distinct subpopulation of less well differentiated hepatocytes. Transplanted immunopurified LAP+ late gestation fetal hepatocytes formed small hepatic, endothelial and occasional ductal colonies within one month. The average size of the colonies derived from the LAP+ cells increased so that by 10 months up to 35% of the liver was repopulated by donor-derived cells. Conclusions: Our studies show that late gestation fetal hepatocytes, despite their being far along in the differentiation process, possess the capacity for extensive liver repopulation. This is likely related to the unexpected presence of a significant proportion of hepatocyte marker-positive cells maintaining a less well differentiated phenotype.
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Project description:Human induced pluripotent stem cells (iPSCs) have the capability of revolutionizing research and therapy of liver diseases by providing a source of hepatocytes for autologous cell therapy and disease modelling. However, despite progress in advancing the differentiation of iPSCs into hepatocytes (iPSC-Heps) in vitro, cells that replicate the ability of human primary adult hepatocytes (aHeps) to proliferate extensively in vivo have not been reported. This deficiency has hampered efforts to recreate human liver diseases in mice, and has cast doubt on the potential of iPSC-Heps for liver cell therapy. The reason is that extensive post-transplant expansion is needed to establish and sustain a therapeutically effective liver cell mass in patients, a lesson learned from clinical trials of aHep transplantation. Here, as a solution to this problem, we report the generation of human fibroblast-derived hepatocytes that can repopulate mouse livers. Unlike current protocols for deriving hepatocytes from human fibroblasts, ours did not generate iPSCs but cut short reprogramming to pluripotency to generate an induced multipotent progenitor cell (iMPC) state from which endoderm progenitor cells and subsequently hepatocytes (iMPC-Heps) could be efficiently differentiated. For this purpose we identified small molecules that aided endoderm and hepatocyte differentiation without compromising proliferation. After transplantation into an immune-deficient mouse model of human liver failure, iMPC-Heps proliferated extensively and acquired levels of hepatocyte function similar to those of aHeps. Unfractionated iMPC-Heps did not form tumours, most probably because they never entered a pluripotent state. Our results establish the feasibility of significant liver repopulation of mice with human hepatocytes generated in vitro, which removes a long-standing roadblock on the path to autologous liver cell therapy.
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Project description:Hepatocyte transplantation is an attractive alternative to liver transplantation. Thus far, however, extensive liver repopulation by adult hepatocytes has required ongoing genetic, physical, or chemical injury to host liver. We hypothesized that providing a regulated proliferative and/or survival advantage to transplanted hepatocytes should enable repopulation in a normal liver microenvironment. Here, we repopulated livers of DPPIV- (dipeptidyl peptidase-4) rats and Ugt1a1 (uridinediphosphoglucuronate glucuronosyltransferase 1a1)-deficient Gunn rats (model of Crigler-Najjar syndrome type 1), both models without underlying liver injury, for up to 1 year by transplanting lenti-hYAP-ERT2 (mutated estrogen receptor ligand-binding domain 2)-transduced hepatocytes (YAP-Hc). Yap (yes-associated protein) nuclear translocation/function in YAP-Hc was regulated by tamoxifen. Repopulating YAP-Hc and host hepatocytes were fluorescence-activated cell sorting-purified and their transcriptomic profiles compared by RNAseq. After 1 year of liver repopulation, YAP-Hc clusters exhibited normal morphology, integration into hepatic plates and hepatocyte-specific gene expression, without dysplasia, dedifferentiation, or tumorigenesis. RNAseq analysis showed up-regulation of 145 genes promoting cell proliferation and 305 genes suppressing apoptosis, including hepatocyte growth factor and connective tissue growth factor among the top 30 in each category and provided insight into the mechanism of cell competition that enabled replacement of host hepatocytes by YAP-Hc. In Gunn rats transplanted with YAP-Hc+tamoxifen, there was a 65%-81% decline in serum bilirubin over 6 months versus 8%-20% with control-Hc, representing a 3-4-fold increase in therapeutic response. This correlated with liver repopulation as demonstrated by the presence of Ugt1a1-positive hepatocyte clusters in livers and western blot analysis of tissue homogenates. Conclusion: Tamoxifen-regulated nuclear translocation/function of hYAP-ERT2 enabled long-term repopulation of DPPIV-/- and Gunn rat livers by hYAP-ERT2-transduced hepatocytes without tumorigenesis. This cell transplantation strategy may offer a potential therapy for most of the inherited monogenic liver diseases that do not exhibit liver injury.
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Project description:Current research focuses on developing alternative strategies to restore decreased liver mass prior to the onset of end-stage liver disease. Cell engraftment/repopulation requires regeneration in normal liver, but we have shown that severe liver injury stimulates repopulation without partial hepatectomy (PH). We have now investigated whether a less severe injury, secondary biliary fibrosis, would drive engraftment/repopulation of ectopically transplanted mature hepatocytes.Ductular proliferation and progressive fibrosis in dipeptidyl-peptidase IV (DPPIV)(-) F344 rats was induced by common bile duct ligation (BDL). Purified DPPIV(+)/green fluorescent protein (GFP)(+) hepatocytes were infused without PH into the spleen of BDL rats and compared to rats without BDL.Within one week, transplanted hepatocytes were detected in hepatic portal areas and at the periphery of expanding portal regions. DPPIV(+)/GFP(+) repopulating cell clusters of different sizes were observed in BDL rats but not untreated normal recipients. Surprisingly, some engrafted hepatocytes formed CK-19/claudin-7 expressing epithelial cells resembling cholangiocytes within repopulating clusters. In addition, substantial numbers of hepatocytes engrafted at the intrasplenic injection site assembled into multicellular groups. These also showed biliary "transdifferentiation" in the majority of intrasplenic injection sites of rats that received BDL but not in untreated recipients. PCR array analysis showed upregulation of osteopontin (SPP1). Cell culture studies demonstrated increased Itg?4, HNF1?, HNF6, Sox-9, and CK-19 mRNA expression in hepatocytes incubated with osteopontin, suggesting that this secreted protein promotes dedifferentiation of hepatocytes.Our studies show that biliary fibrosis stimulates liver repopulation by ectopically transplanted hepatocytes and also stimulates hepatocyte transition towards a biliary epithelial phenotype.
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Project description:ObjectivesSeptic cardiomyopathy develops frequently in patients with sepsis and likely increases short-term mortality. However, whether septic cardiomyopathy is associated with long-term outcomes after sepsis is unknown. We investigated whether septic patients with septic cardiomyopathy have worse long-term outcomes than septic patients without septic cardiomyopathy.DesignRetrospective cohort study.SettingAdult ICU.PatientsAdult ICU patients with sepsis.InterventionsNone.Measurements and main resultsLeft ventricular global longitudinal systolic strain was our primary measure of septic cardiomyopathy. We employed a suite of multivariable survival analyses to explore linear and nonlinear associations between left ventricular global longitudinal systolic strain and major adverse cardiovascular events, which included death, stroke, and myocardial infarction. Our primary outcome was major adverse cardiovascular event through 24 months after ICU discharge. Among 290 study patients, median left ventricular global longitudinal systolic strain was -16.8% (interquartile range, -20.4% to -12.6%), and 38.3% of patients (n = 111) experienced a major adverse cardiovascular event within 24 months after discharge. On our primary, linear analysis, there was a trend (p = 0.08) toward association between left ventricular global longitudinal systolic strain and major adverse cardiovascular event (odds ratio, 1.03; CI, < 1 to 1.07). On our nonlinear analysis, the association was highly significant (p < 0.001) with both high and low left ventricular global longitudinal systolic strain associated with major adverse cardiovascular event among patients with pre-existing cardiac disease. This association was pronounced among patients who were younger (age < 65 yr) and had Charlson Comorbidity Index greater than 5.ConclusionsAmong patients with sepsis and pre-existing cardiac disease who survived to ICU discharge, left ventricular global longitudinal systolic strain demonstrated a U-shaped association with cardiovascular outcomes through 24 months. The relationship was especially strong among younger patients with more comorbidities. These observations are likely of use to design of future trials.
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