Introduction: Hepatocyte-like cells (HLCs) differentiated from human induced pluripotent stem cells (hiPSCs) offer an alternative model for primary human hepatocytes (PHHs) when analyzing the functionality and lipid secretion properties of liver is desired in a patient-specific manner. The detailed lipid profile of HLCs is, however, unknown. Here, we aim to develop an in vitro model of hepatocytes derived from patients with coronary artery disease (CAD) in order to study their lipidomics.
Method: hiPSCs were derived from skin biopsies and then differentiated into functional HLCs. Five hepatic differentiation methods were tested to find the most efficient and robust protocol. During the entire differentiation process from hiPSCs to HLCs, we monitored the detailed quantification of alteration in cellular lipidome and gene expression profiling of a set of 16 lipid metabolism-related genes using mass spectrometry and qPCR respectively. Additionally, biochemical (TG, albumin, and urea secretion), functional (LDL uptake) gene (SOX17, FOXA2, AFP, ALB, APOA1, and APOB), and protein (SOX17, AFP, ALB, ASGPR) expression measurements were done. Furthermore, the transcriptome of HLCs was studied by Agilient human miRNA microarray. All results from HLCs were compared to PHHs and a hepatoma-derived cell line (HepG2).
Results: The lipidome and lipid-related gene expression of the HLCs resemble those of the PHHs, and the HLCs display the expected morphology and cellular functions of a functional hepatocyte. An elevation was observed in the production of very-long chain sphingolipids and polyunsaturated fatty acid-containing phospholipids during the hepatic maturation. We show that cells efficiently take up fatty acids from the media, incorporate them, and modify simple lipids into more complex ones mirrored in their altered cellular functions.
Conclusion: We have successfully set up an in vitro patient specific hepatocyte model which is functional and is capable of both uptaking and secreting lipids. This model can be an invaluable platform which can be applied e.g. when exploring different lipids involved in the development of diseases such as CAD, but the platform can also be utilized in other applications that currently PHHs are employed for.