Abstract
The liver is among the principal organs for glucose homeostasis and metabolism. Studies of liver metabolism are limited by the inability to expand primary hepatocytes in vitro while maintaining their metabolic functions. Human hepatic three-dimensional (3D) organoids have been established using defined factors, yet hepatic organoids from adult donors showed impaired expansion. We examined conditions to facilitate the expansion of adult donor-derived hepatic organoids (HepAOs) and HepG2 cells in organoid cultures (HepGOs) using combinations of growth factors and small molecules. The expansion dynamics, gluconeogenic and HNF4α expression, and albumin secretion are assessed. The conditions tested allow the generation of HepAOs and HepGOs in 3D cultures. Nevertheless, gluconeogenic gene expression varies greatly between conditions. The organoid expansion rates are limited when including the TGFβ inhibitor A8301, while are relatively higher with Forskolin (FSK) and Oncostatin M (OSM). Notably, expanded HepGOs grown in the optimized condition maintain detectable gluconeogenic expression in a spatiotemporal distribution at 8 weeks. We present optimized conditions by limiting A8301 and incorporating FSK and OSM to allow the expansion of HepAOs from adult donors and HepGOs with gluconeogenic competence. These models increase the repertoire of human hepatic cellular tools available for use in liver metabolic assays.
Highlights
Liver diseases account for about two million deaths annually worldwide [1]
When cultured under low glucose concentrations corresponding to normoglycemia (1 g/L), the cell growth of HepG2 cells was not significantly affected, while the growth rate of HuH7 cells was highly reduced in normoglycemic conditions, similar to previous reports [55], suggesting that HepG2 cells could be utilized to optimize the hepatocyte-like and metabolic functions in 3D organoid cultures
Organoids have been derived from tissue-resident adult stem-like cells (ASC), iPSCs, and amniotic cells using different combinations of growth factors and small molecules [30,31,33,34,37,48,62,63,64,65,66]
Summary
Liver diseases account for about two million deaths annually worldwide [1]. Chronic liver diseases are caused mainly by viral infections, excess alcohol intake, fatty liver, autoimmune, and drug-related liver diseases and hepatocellular carcinoma (HCC) [2]. Non-alcoholic fatty liver disease (NAFLD) comprises fatty liver (steatosis), non-alcoholic steatohepatitis (NASH), and fibrosis/cirrhosis and may lead to HCC. Metabolic and physiological shifts in many liver diseases are linked to the gluconeogenic pathway [4,5,6,7]. Insulin resistance initiates liver steatosis by providing substrates for mitochondrial β-oxidation such as glucose and glycerol [8]. This increased gluconeogenesis and elevated liver glucose production contribute to fasting hyperglycemia in T2DM patients [8]. The effects of hepatic cell culture conditions on glucose production and gluconeogenic functions have not been well-characterized
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