Dedifferentiation of Primary Hepatocytes is Accompanied with Reorganization of Lipid Metabolism Indicated by Altered Molecular Lipid and miRNA Profiles

• Main Authors: Mostafa Kiamehr, Laura Heiskanen, Thomas Laufer, Aneta Düsterloh, Mustafa Kahraman, Reijo Käkelä, Reijo Laaksonen, Katriina Aalto-Setälä
• Format: Article
• Language: English
• Published: MDPI AG 2019-06-01
• Series: International Journal of Molecular Sciences
• Subjects: primary human hepatocytes (PHHs); dedifferentiation; lipidomics; mass spectrometry; sphingolipids (SLs); phospholipids (PLs); saturated fatty acids (SFAs); monounsaturated fatty acids (MUFAs); polyunsaturated fatty acids (PUFAs); microRNAs (miRNAs)
• Online Access: https://www.mdpi.com/1422-0067/20/12/2910

Aim: Primary human hepatocytes (PHHs) undergo dedifferentiation upon the two-dimensional (2D) culture, which particularly hinders their utility in long-term in vitro studies. Lipids, as a major class of biomolecules, play crucial roles in cellular energy storage, structure, and signaling. Here, for the first time, we mapped the alterations in the lipid profile of the dedifferentiating PHHs and studied the possible role of lipids in the loss of the phenotype of PHHs. Simultaneously, differentially expressed miRNAs associated with changes in the lipids and fatty acids (FAs) of the dedifferentiating PHHs were investigated. Methods: PHHs were cultured in monolayer and their phenotype was monitored morphologically, genetically, and biochemically for five days. The lipid and miRNA profile of the PHHs were analyzed by mass spectrometry and Agilent microarray, respectively. In addition, 24 key genes involved in the metabolism of lipids and FAs were investigated by qPCR. Results: The typical morphology of PHHs was lost from day 3 onward. Additionally, <i>ALB</i> and <i>CYP</i> genes were downregulated in the cultured PHHs. Lipidomics revealed a clear increase in the saturated fatty acids (SFA) and monounsaturated fatty acids (MUFA) containing lipids, but a decrease in the polyunsaturated fatty acids (PUFA) containing lipids during the dedifferentiation of PHHs. In line with this, <i>FASN</i>, <i>SCD</i>, <i>ELOVL1</i>, <i>ELOVL3</i>, and <i>ELOVL7</i> were upregulated but <i>ELOVL2</i> was downregulated in the dedifferentiated PHHs. Furthermore, differentially expressed miRNAs were identified, and the constantly upregulated miR-27a and miR-21, and downregulated miR-30 may have regulated the synthesis, accumulation and secretion of PHH lipids during the dedifferentiation. Conclusion: Our results showed major alterations in the molecular lipid species profiles, lipid-metabolizing enzyme expression as wells as miRNA profiles of the PHHs during their prolonged culture, which in concert could play important roles in the PHHs&#8217; loss of phenotype. These findings promote the understanding from the dedifferentiation process and could help in developing optimal culture conditions, which better meet the needs of the PHHs and support their original phenotype.