Establishment and characterization of several liver cell lines as tools for the study of physio-pathological cellular interplay

Establishment and characterization of cell lines as tools for the study of physio-pathological liver cellular interplay The liver is the largest internal organ of the body, constituting approximately 2% to 5% of body weight in the adult and 5% in the neonate. This organ plays a central role in metabolic homeostasis and it is responsible for the synthesis, storage and redistribution of nutrients, carbohydrates, fats and vitamins. The liver has a peculiar and fascinating ability: it is able to regenerate itself after loss of parenchyma for surgical resection or injury caused by drugs, toxins or acute viral disease. Considering the variety of liver functions, it is not surprising that a large number of cell types and cell–cell interactions are required for its functionality. Most of the liver functions are carried out by the hepatocytes (about 70-75% of hepatic cells); these, together with cholangiocytes (10-5 %), both of endodermal derivation, constitute the hepatic parenchyma. The other 20% made up of non-parenchymal cells, includes: 1) Kupffer cells, essential for the phagocytosis of foreign particles as well as for the cytokines production, 2) stellate cells, that store vitamin A and produce extra-cellular matrix (ECM) components, 3) sinusoidal endothelial cells, that line the hepatic sinusoids providing a large surface for nutrients absorption and 4) lymphocytes, that mediate adaptive immune responses. A unique architectural arrangement of hepatic parenchymal and non-parenchymal cells governs liver functionality. The need to regenerate functional liver tissue in vitro is well established in three areas of application: model tissue for drug testing, bio artificial liver supports, and potentially engineered organs for implantation into patients. The ability of the mammalian liver to regenerate in vivo indicates that within correct stimulatory environment it should be possible to grow large quantities of liver tissue in vitro. Our interest is always been to understand the minimal environmental signals that are required to generate liver tissue that is able to perform a physiological cellular interplay with parenchymal and non-parenchymal cells. For these reasons, we performed different cellular models. We previously described the identification and characterization of an immortalized bipotential precursor cell within the MMH lines. MMHs (from Met murine hepatocyte) are immortalized and untransformed cell lines derived from explants of liver derived from transgenic mice expressing a constitutively active truncated human Met receptor (cyto-Met) under control of the human 1-antitrypsin transcription unit. MMH lines present an epithelial cell polarity and the expression of hepatic functions. We used the MMHs model for the study: Hepatocyte transformation; Liver differentiation and transdifferentiation (EMT); Cholesterol metabolism; Retinol binding protein regulated secretions; Hemopoiesis and Hepatic influence of HSC. In addition, we previously isolated characterized a number of stable liver stem cell lines named RLSCs (from resident liver stem cells) that spontaneously acquire an epithelial morphology and differentiate into hepatocyte named RLSCdH (from RLSC derived Hepatocytes). Thanks to this model we understood the hepatocyte post-differentiative patterning define “zonation”: their spontaneous differentiation, in fact, generates periportal hepatocytes that may be induced to switch into perivenular hepatocytes by means of the convergence of Wnt signalling on the HNF4-driven transcription. Recently, we demonstrate that RLSCs are able to differentiate in vivo (orthotopic transplants and heterotopic transplants) in epithelial and mesenchymal derivatives. Our data suggesting, for the first time, the existence of an adult stem/precursor cell capable of providing both parenchymal and non-parenchymal components to a complex epithelial organ. For the study of environment signals necessary to liver regeneration in vitro, is necessary the presence of sinusoidal endothelial cellular model. In fact, liver endothelium is a prime example of organ-specific microvascular differentiation and functions. The liver sinusoidal endothelial (LSECs) cells are a morphologically and functionally unique sub-population of liver endothelial cells that form the lining of the hepatic sinusoids. They possess fenestrations that are approximately 50–150 nm in diameter and most are aggregated into groups of 10–100, so-called liver sieve plates. The diameter and number of fenestrations are altered by various liver diseases, diabetes mellitus and old age and are influenced by cytokines and hormones . Alteration in the size and number of fenestrations influences the hepatic trafficking of lipoproteins , clearance of pharmaceutical agents , liver regeneration and interactions between lymphocytes and hepatocytes. Decapillarization is a dedifferentiation process that occurs in vitro over time with SEC in culture. Determinants of endothelial cell phenotype include heterotypic contact with pericytes or smooth muscle cells, paracrine effects of epithelial cells, shear stress, and the underlying substratum. Little is known about the determinants of the normal SEC phenotype. Proximity to liver tissue can induce the SEC phenotype, but the pathways that regulate this have not been established. This is because endothelial primary cells are generally difficult and time–consuming to isolate, limited in number, invariably contain impurities with other cell types, and may lack the features of pathologic vasculature. For these reasons, many authors performed different immortalization strategies on freshly isolated SECs (immortalization with SV40 Middle T or Large T antigens) to obtain a cellular model for the study endothelial-specific functions. Nevertheless these approaches can induce activation of endothelial- specific pathways in aberrant way. Object The current study is aimed to obtain an sinusoidal endothelial cell line as in vitro system for the identification of critical microenvironment factors involved in liver cellular interplay during physiology or pathology conditions. We hypothesis to improve protocols of liver tissue engineering using the our cellular models. Results The current study to go beyond the limits for maintaining LSECs in vitro, thanks the effects of soluble factors released by MMH lines (“Met Murine Hepatocytes” cells are immortalised and untransformed hepatocyte) on the sinusoidal endothelial cells phenotype in vitro and in vivo. Thanks hapatocytes soluble factors, we are able to obtain a “spontaneously immortalized murine liver sinusoidal endothelial cells line”. In fact, Hepatocyte Conditioned medium was able to promote a substantial expansion of sinusoidal endothelial cells and their differentiation phenotype. In Orthotopic transplants MLECs show an intrinsic capability to organize in physiological way and are able to maintain a differentiation state. Also, MLECs are able to promote cellular interplay between parenchymal and non-parenchymal cells, as shown by a sub-endothelial localization of pericyte, in proximity of sinusoids performed by MLECs. In vitro studies showed that MLECs cultured with hepatocyte soluble factors, maintained the greatest degree of differentiation, as showed by high levels of endothelial cells markers expression, such as CD105, CD144, MECA32, VEGFR2. Also, MLECs are able to organize in tube-like structures on matrigel-coat as in vivo. It’s induced that the maintenance of cell phenotype is dependent on micro environmental signals such as paracrine interactions (cell–cell), physical-chemical factors (oxygen tension, metabolites), mechanical stimuli, and cell–extracellular matrix (ECM) interaction. Highlights these data suggest that MMH, RLSCs and MLECs are models that permit: -the study of molecular bases of cellular interplay in physiology and pathology; -to improve protocols of liver tissue engineering.