Microbial‐derived lithocholic acid and vitamin K2 drive the metabolic maturation of pluripotent stem cells–derived and fetal hepatocytes

Abstract

The liver is the main organ responsible for the modification, clearance, and transformational toxicity of most xenobiotics owing to its abundance in cytochrome P450 (CYP450) enzymes. However, the scarcity and variability of primary hepatocytes currently limits their utility. Human pluripotent stem cells (hPSCs) represent an excellent source of differentiated hepatocytes; however, current protocols still produce fetal-like hepatocytes with limited mature function. Interestingly, fetal hepatocytes acquire mature CYP450 expression only postpartum, suggesting that nutritional cues may drive hepatic maturation. We show that vitamin K 2 and lithocholic acid, a by-product of intestinal flora, activate pregnane X receptor (PXR) and subsequent CYP3A4 and CYP2C9 expression in hPSC-derived and isolated fetal hepatocytes. Differentiated cells produce albumin and apolipoprotein B100 at levels equivalent to primary human hepatocytes, while demonstrating an 8-fold induction of CYP450 activity in response to aryl hydrocarbon receptor (AhR) agonist omeprazole and a 10-fold induction in response to PXR agonist rifampicin. Flow cytometry showed that over 83% of cells were albumin and hepatocyte nuclear factor 4 alpha (HNF4a) positive, permitting high-content screening in a 96-well plate format. Analysis of 12 compounds showed an R 2 correlation of 0.94 between TC50 values obtained in stem cellderived hepatocytes and primary cells, compared to 0.62 for HepG2 cells. Finally, stem cell-derived hepatocytes demonstrate all toxicological endpoints examined, including steatosis, apoptosis, and cholestasis, when exposed to nine known hepatotoxins. Conclusion: Our work provides fresh insights into liver development, suggesting that microbial-derived cues may drive the maturation of CYP450 enzymes postpartum. Addition of these cues results in the first functional, inducible, hPSC-derived hepatocyte for predictive toxicology. (HEPATOLOGY 2015;62:265-278) T he liver is the largest internal organ in the human body, responsible for protein synthesis and metabolic homeostasis. Metabolic transformation of lipids is primarily carried out by the cytochrome P450 (CYP450) family of monooxygenases, also responsible for xenobiotic transformation.

Pre- and post-weaning diet alters the faecal metagenome in the cat with differences in vitamin and carbohydrate metabolism gene abundances

“…Although vitamins can be obtained from a variety of foods, some vitamins (e.g., vitamin B1 (thiamine), vitamin B9 (folic acid) and vitamin B12 (cobalamin)) can also be provided through microbial de novo biosynthesis1112. Indeed, microbe-derived vitamins may be of particular importance when diets are deficient in these vitamins.…”

Connexin 32-mediated cell-cell communication is essential for hepatic differentiation from human embryonic stem cells

“…With pluripotency and unlimited self-renewal capacity, human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) could become an effective source of human hepatocytes for clinical and industrial applications5678. Deriving of hepatocytes from either hESCs or hiPSCs have been attempted numerously until now691011. However, hESC/iPSC-derived hepatocytes usually carried fetal hepatocyte-like phenotype and functions12, which indicated that maturation of these derived hepatic cells in vitro was a popular problem and one of the major challenges in research.…”

“…Hepatic progenitor cells were indeed repeatedly found to switch from Cx43 to Cx26 expression and, in particular, to Cx32 expression upon differentiation into hepatocytes, both in vivo 223031 and in vitro 3132. The natural compound Vitamin K2 (VK2) from the gut microbiome has been reported to contribute to the maturation of pluripotent stem cells to hepatocytes10. Previous data suggested that VK2 could up-regulate Cx32 at the transcriptional level33.…”

Innovation for hepatotoxicity in vitro research models: A review

“…With the help of vitamin K2 and lithocholic acid, over 83% of iPSCs/HLCs were positive for the protein of albumin and HNF4α. iPSCs/HLCs produced albumin and apolipoprotein B100 at levels equivalent to those in human PHs, while demonstrating an eightfold induction of CYP450 activity in response to the aryl hydrocarbon receptor agonist omeprazole and a 10‐fold induction in response to PXR agonist rifampicin (Avior et al, ). The cell‐cycle regulator p53 has been shown to act as a critical barrier to the reprogramming process of iPSC/HLCs by inhibiting cell proliferation and promoting apoptosis, and knockout of the TP53 gene in mouse and human fibroblasts produced significantly more iPSC colonies.…”