Large‐Scale Production of LGR5‐Positive Bipotential Human Liver Stem Cells

Abstract

Background and Aims The gap between patients on transplant waiting lists and available donor organs is steadily increasing. Human organoids derived from leucine‐rich repeat‐containing G protein‐coupled receptor 5 (LGR5)–positive adult stem cells represent an exciting new cell source for liver regeneration; however, culturing large numbers of organoids with current protocols is tedious and the level of hepatic differentiation is limited. Approach and Results Here, we established a method for the expansion of large quantities of human liver organoids in spinner flasks. Due to improved oxygenation in the spinner flasks, organoids rapidly proliferated and reached an average 40‐fold cell expansion after 2 weeks, compared with 6‐fold expansion in static cultures. The organoids repopulated decellularized liver discs and formed liver‐like tissue. After differentiation in spinner flasks, mature hepatocyte markers were highly up‐regulated compared with static organoid cultures, and cytochrome p450 activity reached levels equivalent to hepatocytes. Conclusions We established a highly efficient method for culturing large numbers of LGR5‐positive stem cells in the form of organoids, which paves the way for the application of organoids for tissue engineering and liver transplantation.

Human extrahepatic and intrahepatic cholangiocyte organoids show region-specific differentiation potential and model cystic fibrosis-related bile duct disease

“…Comparative gene expression analysis of EM and DM-hep of 35 genes specific for hepatocytes, cholangiocytes or progenitor cells selected from the human liver atlas 15 is shown in Fig. 5 B. Gene expression of primary hepatocytes (PHH) as published by Schneeberger et al 22 were included as a benchmark for hepatocyte-fate differentiation. Expression of the hepatocyte-specific genes were clearly different between ICO and ECO.…”

Radiation‐induced tissue damage and response

“…Like the kidney, the liver has low turnover but can be stimulated to regenerate rapidly after surgery, leading to the general assumption that all mature hepatocytes are able to maintain homeostasis. However, lineage tracing in mice using Wnt-responsive Axin2 identified a population of proliferating and self-renewing diploid cells adjacent to the central vein in the liver lobule that could give rise to mature polyploid hepatocytes [102] and LGR5 + adult liver stem cells can be grown as organoids [103]. The origin of the liver clones that grow on transplantation to recipient mice and whose radiation characteristics have been examined and found to be characteristic of a late responding tissue, is not known [23,104].…”

Scaffolds obtained from decellularized human extrahepatic bile ducts support organoids to establish functional biliary tissue in a dish

“…The cells that give rise to these biliary organoids are EPCAM positive and organoids from all three sources maintain cholangiocyte‐specific markers (e.g., EPCAM, cytokeratin 7 [KRT‐7] or cytokeratin 19 [KRT‐19]) and functionality (Aizarani et al, 2019; Aloia et al, 2019; Huch et al, 2015; Rimland et al, 2020; Sampaziotis et al, 2017; Soroka et al, 2018). Furthermore, relative small biopsies (0.5–1.0 g tissue or 1 ml of bile) are adequate to initiate cultures, which subsequently can yield millions of cells (Schneeberger et al, 2020; Willemse et al, 2017). These characteristics make the organoids ideal cell sources for the repopulation of decellularized EBD scaffolds in an effort to create functional tissue engineered bile ducts in vitro.…”