Human liver model systems in a dish

Abstract

The human adult liver has a multi-cellular structure consisting of large lobes subdivided into lobules containing portal triads and hepatic cords lined by specialized blood vessels. Vital hepatic functions include filtering blood, metabolizing drugs, and production of bile and blood plasma proteins like albumin, among many other functions, which are generally dependent on the location or zone in which the hepatocyte resides in the liver. Due to the liver's intricate structure, there are many challenges to design differentiation protocols to generate more mature functional hepatocytes from human stem cells and maintain the long-term viability and functionality of primary hepatocytes. To this end, recent advancements in three-dimensional (3D) stem cell culture have accelerated the generation of a human miniature liver system, also known as liver organoids, with polarized epithelial cells, supportive cell types and extra-cellular matrix deposition by translating knowledge gained in studies of animal organogenesis and regeneration. To facilitate the efforts to study human development and disease using in vitro hepatic models, a thorough understanding of state-of-art protocols and underlying rationales is essential. Here, we review rapidly evolving 3D liver models, mainly focusing on organoid models differentiated from human cells.

Keywords: liver; organogenesis; organoid; regeneration; stem cell.

Figure 1.. Schematic of liver bud formation.

Liver progenitors are first specified from the ventral foregut endoderm which is surrounded by the STM and endothelial cells that supply necessary factors. The hepatoblast cell layer thickens and begins to delaminate from the epithelium and bud out through the STM to form the liver bud. The liver bud then undergoes growth and maturation throughout development to form the fetal liver. (abbreviations: septum transversum mesenchyme (STM), endothelial cells (EC), hepatic endoderm (HE)).

Figure 2.. Research interest and publications for liver organoids has increased greatly over the past 5 years.

A pubmed search using the query (liver OR hepatic) AND (organoid [title] OR organ bud [title]) demonstrates a continued increase in liver organoid papers.

Figure 3.. A. Examples of step-wise directed differentiation of 3D hPSC derived hepatic organoids or spheroids.

Nearly all the hPSC derived hepatic 3D models first undergo a DE stage before modifications are made to the standard hepatic endoderm, hepatoblast and hepatocyte-like cell sequence. *3D: this differentiation starts out as embryoid bodies in suspension culture. B. Markers that correspond to each stage of organoid development. (abbreviations: Posterior foregut (Pfg), Hepatic endoderm (HE), Hepatoblast (HB), Foregut (Fg), Intermediate Hepatoblast (IH), Hepatocytes (Hep), Cholangiocytes (Chol), Stellate cells (Stel), Kupffer cells (Kupf), Dexamethasone (Dex), Epidermal Growth Gactor (EGF), N6,2’-ODibutyryladenosine 3’,5’-cyclic 35 monophosphate sodium salt (dbCAMP), Dickkopf-related protein 1 (DKK-1), Wnt inhibitory factor 1 (WIF-1), Retinoic Acid (RA), Lithocholic Acid (LCA), Vitamin K2 (VK2), Valproic Acid (VPA), N-acetylcysteine (N-ace), Pancreatic and Duodenal Homeobox 1 (PDX1), and Hematopoietically-expressed homeobox (HHEX)).

Figure 4:. Two sources of human liver organoids include cells taken directly from the human liver as well as cells differentiated from hPSCs.

Both can use cells derived from healthy or diseased patients, and hPSCs can also be genetically modified before differentiation. Cells taken from the liver include primary hepatocytes as well as ductal cells and primary hepatocytes can be mixed with other supporting cell types. hPSCs can be co-differentiated in a dish to hepatocytes and cholangiocytes, or hepatocytes and stellate and Kupffer cells, and hepatocytes can also be mixed with other supporting cell types generated from hPSCs.

Figure 5:. Framing next generation organoid systems.

On an inter-cellular scale there can be mixed cell types in one model generated from defined lineages. On an inter-tissue scale supportive tissues are incorporated including vascular, immune or mesenchymal lineages, and there is microanatomical remembrance. An inter-organ scale will include neighboring organ connectivity and macro-anatomical remembrance, while an inter-system scale can have connectivity between distant organs.