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  • All these restrictions called for


    All these restrictions called for the development of better and more efficient human experimental models in vitro, and are at the basis of the newly available technology of induced pluripotent stem Thapsigargin (iPSCs) and the efforts in generating several protocols for their differentiation into liver cells. In addition to iPSCs, the recent discovery of 3D organoids that can be generated from adult liver cells offers another approach to model liver disease in vitro. In this review, we will discuss how iPSC and organoid technologies can be applied to the study of liver pathobiology, with their pros and flaws.
    Liver development in a dish: iPSC Most of the genes and pathways involved in the process of liver development have been elucidated in the last decade through the use of animal models, in particular transgenic mice [[6], [7], [8], [9]]. These notions have recently been used in the lab to generate liver cells in a “dish”. The liver originates from the endoderm, one of the three germ layers formed during embryonic development [10]. Cells of the endoderm are committed to a specific differentiation fate as a consequence of distinct transcription factor expression [11]. In the mouse, around embryonic day E8.25, endoderm cells receive specific extracellular signals that induce their specification to the hepatic fate. A necessary player in this process is FGF [12]. BMP-2 and BMP-4 cooperate with FGF during the hepatic specification [13]. After hepatic endoderm, the next step is the specification of the liver diverticulum lined by endoderm cells called hepatoblasts. This happens at E9 in the mouse embryo and at day 22 in humans. Hepatoblasts proliferate forming a tissue bud and subsequently migrate away from the endoderm epithelium invading the septum transversum where they keep proliferating with further expansion of the liver [10]. A complex network of growth factors is active during this process and is involved in hepatoblasts proliferation. Some of the major components of this network are HGF, TGF-β and FGF10 that are essential for hepatoblasts proliferation [14]. Wnt-signaling (WNT) and β-catenin are also involved during liver development, even though WNT signaling is very complex and has a different role depending on the developmental stage. In early stages of development, WNT signaling has to be repressed anteriorly to preserve foregut identity and promote liver specification, while in the later stages of development WNT is necessary to promote hepatoblasts proliferation and biliary differentiation [15]. Interestingly, retinoic acid signaling is important for liver bud growth and hepatoblasts proliferation [16]. This signaling is regulated by the zinc finger transcription factor Wilms' tumor suppressor gene (WT1), expressed in mesodermal cells [16]. Once hepatoblasts have reached a certain developmental stage they acquire the potential to differentiate either into the hepatocyte or cholangiocyte lineage [17]. This process is directed by the NOTCH signaling pathway through interaction with its ligand JAGGED, present on mesenchymal cells in the periportal areas [18]. NOTCH activation inhibits hepatoblast differentiation into hepatocytes, but propels their differentiation into cholangiocytes inducing expression of SOX9, one of the most specific markers of biliary cells [19]. Following the lineage segregation stage, hapatocytes and cholangiocytes go through a process of maturation with the acquisition of a specific morphology and physiologic function that will continue until after birth [7,10,17]. The advance in stem cell technology has resulted in the ability to reprogram somatic cells into pluripotent stem cells (iPSC), thus providing a unique platform for manufacturing cells from patients with different genetic diseases [20,21]. Using iPSCs makes it possible to recapitulate in a dish the developmental steps leading to a specific tissue, by sequentially adding to the culture a cocktail of growth factors that correspond to the pathways active in vivo as described above for the liver. Moreover, iPSCs can be easily derived from skin fibroblasts or most recently from mononuclear white blood cells or urine samples and in theory, be then differentiated into a variety of cell types [22,23]. Their limitless ability to self-renew and their differentiation potential, allow the production of large amounts of specific cell types affected for example by a genetic disease, or perform gene editing protocols. As iPSCs retain the same genetic background of their donors, they provide an attractive alternative for disease model and drug discovery when the traditional models are inadequate [20]. In the next section, we will describe the different strategies used to induce differentiation of iPSCs into liver cells.