Fatty Acids and Microbial-Derived Cis-9, Trans-11 Conjugated Linoleic Acid Drive the Metabolic and Mitochondrial Maturation of Pluripotent Stem Cells-Derived and Fetal Hepatocytes

Muneef Ayyash 1,6 Avner Ehrlich 1,2 Michal Zimerman 1 Robert Schwartz 4 Idit Goldfracht 3 Matan Hofree 5 Yishai Avior 1 Gahl Levy 1 Merav Cohen 1,2 Daniel Kitsberg 1,2 Lior Gepstein 3 Yaakov Nahmias 1,2,6
1Grass Center for Bioengineering, Israel
2Silberman Institute of Life Sciences, Israel
3Technion- Israel Institute of Technology, Israel
4Weill Cornell Medical College, USA
5Broad Institute of MIT and Harvard, USA

Background: PSC-derived hepatocytes are now considered a reliable cellular alternative for drug development and clinical applications. We have previously shown that post-partum microbial-derived cues, lithocholic acid, and vitamin K2, can drive the metabolic maturation of hESC-derived and fetal hepatocytes by activating PXR. Results have led us to hypothesize about the role of different postpartum nutritional cues on hepatic development and maturation, with special emphasis on mitochondrial development.

Methods: We report a four-step 16-day differentiation protocol to produce a homogenous culture of hPSC-derived hepatocytes.

Results: Mimicking the postnatal nutritional shift, by adding Oleic acid (OA) and Linoleic acid (LA) to the last stage of PSC differentiation, induced a dose-dependent activation of key hepatic nuclear receptors, PPAR, PXR and LXR, essential for the metabolic functionality of the mature hepatocyte. Further supporting the fatty acid-induced maturation, albumin expression increased in a PPAR dependent manner concurrently with a decrease in AFP expression. Replacing LA with microbial-derived 9CLA promoted an additional PPAR-dependent PXR activation, increasing nuclear localization by 30% and up-regulating CYP450 gene expression and activity. Functional and morphological analyses performed to evaluate the influence of fatty acids, showed a PPAR dependent increase in mitochondrial function, biogenesis, and fusion, recapitulating the postnatal developmental processes.

Conclusion: This work provides fresh insights into the role of postnatal nutritional cues in hepatic maturation and mitochondrial development via the activation of lipid-regulated PPAR. Our work sheds the light on the tight link between nutrition, gut colonization and cellular developmental processes that underlie haptic maturation.

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