Keratinocyte serum-free medium maintains long-term liver gene expression and function in cultured rat hepatocytes by preventing the loss of liver-enriched transcription factors

Abstract

Freshly isolated hepatocytes rapidly lose their differentiated properties when placed in culture. Therefore, production of a simple culture system for maintaining the phenotype of hepatocytes in culture would greatly facilitate their study. Our aim was to identify conditions that could maintain the differentiated properties of hepatocytes for up to 28 days of culture. Adult rat hepatocytes were isolated and attached in Williams’ medium E containing 10% serum. The medium was changed to either fresh Williams’ medium E or keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract. The hepatic phenotype was then analysed using RT-PCR, immunohistochemistry, Western blotting and assays of liver function. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their phenotype for 3–4 weeks, based on expression of liver proteins, ureagensis and response to xenobiotics. In contrast, hepatocytes cultured in Williams’ medium E rapidly lost the expression of liver proteins after 3 days. Cells cultured in keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract maintained their expression of liver-enriched transcription factors (C/EBPα and β, HNF4α and RXRα) while expression was either lost or reduced in cells cultured in Williams’ medium E. These results suggest that keratinocyte serum-free medium supplemented with dexamethasone, epidermal growth factor and pituitary gland extract can maintain the hepatic phenotype for a prolonged period and that this is probably related to the continued expression of the liver-enriched transcription factors.

Direct Lineage Conversion of Terminally Differentiated Hepatocytes to Functional Neurons

“…20%) (Figure 3H and supplementary methods). When cultured in keratinocyte serum free media (KSFM), a media reported to prevent de-differentiation of cultured hepatocytes (Li et al, 2007) we observed conversion efficiencies similar to our regular hepatocyte growth media (Figure S1E,F). …”

Hydrogel embedding of precision‐cut liver slices in a microfluidic device improves drug metabolic activity

“…This indicated that the decrease was independent of the incubation system, provided slices did not adhere to the ceiling of the microchamber. Various medium supplements have been shown to improve the maintenance of the metabolic rate for longer periods of time (Agius et al, 1986;Li et al, 2007). It was therefore decided to supplement the medium with 5% v/v heat-inactivated calf serum, as has commonly been done by others when liver slices were incubated longer than 48 h (De Graaf et al, 2007).…”

Efficient programming of human mesenchymal stem cell‐derived hepatocytes by epigenetic regulations

“…Because the shortage of viable liver organs available for transplant remains an issue, the use of hepatocyte transplantation and artificial liver devices as alternative therapeutic approaches has been proposed . However, the limited lifespan and proliferative capacity as well as rapid de‐differentiation after hepatocyte isolation may hinder the usefulness of the in vitro hepatocytes for liver transplantation . Recent studies suggested that human embryonic or tissue‐specific adult stem cells could be directed to become functional hepatocyte‐like cells for clinical applications and drug development, although optimal differentiation protocol remained to be determined …”

“…1 However, the limited lifespan and proliferative capacity as well as rapid de-differentiation after hepatocyte isolation may hinder the usefulness of the in vitro hepatocytes for liver transplantation. 2,3 Recent studies suggested that human embryonic or tissue-specific adult stem cells could be directed to become functional hepatocyte-like cells for clinical applications and drug development, although optimal differentiation protocol remained to be determined. 4 Human mesenchymal stem (stromal) cells (hMSCs) are currently considered a key cell source for tissue engineering because of their relatively easy access and wider distribution in different parts of the body and great expansion and differentiation potential into various cell lineages.…”