Identification of the molecular events underlying liver stem cell choices at the branch of epithelial/mesenchymal differentiation

Beside the many promises and expectation, as yet current therapeutic applications of tissue engineering and regenerative medicine are restricted to a limited number of epithelial tissues (i.e. skin, cornea and heart valve) [1] where both 3D structure and instructive signals arising from cellular interplay show a low level of complexity. Regarding the liver, the challenge of the use of tissue engineering for the treatment of acute and chronic hepatic failure is still far from won; the organ, in fact, is composed by several histotypes that guarantee liver functions only when they correctly set up cell-cell interactions in the complex anatomical architecture of the hepatic lobule. Moreover, local micro-environmental elements, such as growth factors and both stiffness and composition of the extracellular matrix (ECM), provide important cues to determine cell fate. Particularly, mechanical properties of the ECM play a pivotal role in the regulation of many important cell processes that, eventually, decide cell fate and function. Here, we present experimental data obtained with the use of a new cellular tool constituted by resident liver stem cells (RLSCs), derived from murine livers and established in lines [2]. These cells display a metastable phenotype, as underlined by i) the co-expression of epithelial and mesenchymal markers and ii) the ability to differentiate toward two mutually exclusive epithelial or mesenchymal derivatives: hepatocytes and hepatic stellate cells (HSCs). These progenitor cells, indeed, when transplanted in healthy growing livers (i.e. in murine newborns 1-2 days old) were found to contribute to the liver tissue with both hepatocytes and HSCs properly integrated in the hepatic architecture. Heterotopic transplantations, while confirm the dual differentiation potentiality of RLSCs, indicate as tissue local cues are necessary to drive a full hepatic differentiation [3]. One of the local elements mainly influencing the liver cell function is the elasticity/stiffness of the ECM. Liver tissue in physiological condition displays a liver stiffness (LS) lower than 6 KPa. LS increases during progression of liver fibrosis, a pathological state shared by several liver diseases and characterized by proliferation of collagen producing cells [4]. Our results showed that mechanical stimuli drive RLSCs differentiation choices within 24 hours: soft matrix (0,4 KPa) selectively induces epithelial differentiation while stiff matrix (80 KPa) induces mesenchymal differentiation. We are currently characterizing, in this dynamic cellular model, the epigenetic events that anticipate liver-specific gene expression, focusing in particular on the promoter region of HNF4alpha, a transcriptional master regulator of hepatic differentiation. Our data would disclose new insight into the early events driving pivotal cellular choices at the branch of epithelial/mesenchymal differentiation.