hiPSC-derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver.

Hepatocyte-like cells (HLCs) differentiated from human-induced pluripotent stem cells offer an alternative platform to primary human hepatocytes (PHHs) for studying the lipid metabolism of the liver. However, despite their great potential, the lipid profile of HLCs has not yet been characterized. Here, we comprehensively studied the lipid profile and fatty acid (FA) metabolism of HLCs and compared them with the current standard hepatocyte models: HepG2 cells and PHHs. We differentiated HLCs by five commonly used methods from three cell lines and thoroughly characterized them by gene and protein expression. HLCs generated by each method were assessed for their functionality and the ability to synthesize, elongate, and desaturate FAs. In addition, lipid and FA profiles of HLCs were investigated by both mass spectrometry and gas chromatography and then compared with the profiles of PHHs and HepG2 cells. HLCs resembled PHHs by expressing hepatic markers: secreting albumin, lipoprotein particles, and urea, and demonstrating similarities in their lipid and FA profile. Unlike HepG2 cells, HLCs contained low levels of lysophospholipids similar to the content of PHHs. Furthermore, HLCs were able to efficiently use the exogenous FAs available in their medium and simultaneously modify simple lipids into more complex ones to fulfill their needs. In addition, we propose that increasing the polyunsaturated FA supply of the culture medium may positively affect the lipid profile and functionality of HLCs. In conclusion, our data showed that HLCs provide a functional and relevant model to investigate human lipid homeostasis at both molecular and cellular levels.

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

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, ALB and CYP 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, FASN, SCD, ELOVL1, ELOVL3, and ELOVL7 were upregulated but ELOVL2 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' 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.

Long-term performance and stability of molecular shotgun lipidomic analysis of human plasma samples.

The stability of the lipid concentration levels in shotgun lipidomics analysis was tracked over a period of 3.5 years. Concentration levels in several lipid classes, such as phospholipids, were determined in human plasma lipid extracts. Impact of the following factors on the analysis was investigated: sample amount, internal standard amount, and sample dilution factor. Moreover, the reproducibility of lipid profiles obtained in both polarity modes was evaluated. Total number of samples analyzed was approximately 6800 and 7300 samples in negative and positive ion modes, respectively, out of which 610 and 639 instrument control samples were used in stability calculations. The assessed shotgun lipidomics approach showed to be remarkably robust and reproducible, requiring no batch corrections. Coefficients of variation (CVs) of lipid mean concentration measured with optimized analytical parameters were typically less than 15%. The high reproducibility indicated that no lipid degradation occurred during the monitored time period.

High-grade ovarian serous carcinoma patients exhibit profound alterations in lipid metabolism.

Ovarian cancer is a very severe type of disease with poor prognosis. Treatment of ovarian cancer is challenging because of the lack of tests for early detection and effective therapeutic targets. Thus, new biomarkers are needed for both diagnostics and better understanding of the cellular processes of the disease. Small molecules, consisting of metabolites or lipids, have shown emerging potential for ovarian cancer diagnostics. Here we performed comprehensive lipidomic profiling of serum and tumor tissue samples from high-grade serous ovarian cancer patients to find lipids that were altered due to cancer and also associated with progression of the disease. Ovarian cancer patients exhibited an overall reduction of most lipid classes in their serum as compared to a control group. Despite the overall reduction, there were also specific lipids showing elevation, and especially alterations in ceramide and triacylglycerol lipid species were dependent on their fatty acyl side chain composition. Several lipids showed progressive alterations in patients with more advanced disease and poorer overall survival, and outperformed CA-125 as prognostic markers. The abundance of many serum lipids correlated with their abundance in tumor tissue samples. Furthermore, we found a negative correlation of serum lipids with 3-hydroxybutyric acid, suggesting an association between decreased lipid levels and fatty acid oxidation. In conclusion, here we present a comprehensive analysis of lipid metabolism alterations in ovarian cancer patients, with clinical implications.