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

Received: 19 April 2018 | Accepted: 9 July 2018 DOI: 10.1002/jcp.27131 ORIGINAL RESEARCH ARTICLE hiPSC‐derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver Mostafa Kiamehr 1 | Anna Alexanova 1 | Leena E. Viiri 1 | Laura Heiskanen 2 | Terhi Vihervaara 2 | Dimple Kauhanen 2 | Kim Ekroos 5 | Reijo Laaksonen 1,2 | Reijo Käkelä 3 | Katriina Aalto‐Setälä 1,4 1 Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland 2 Zora Biosciences, Espoo, Finland 3 Faculty of Biology and Environmental Sciences, University of Helsinki, Helsinki, Finland 4 Heart Hospital, Tampere University Hospital, Tampere, Finland 5 Lipidomics Consulting Ltd, Espoo, Finland Correspondence Mostafa Kiamehr, Faculty of Medicine and Life Sciences, University of Tampere, Tampere, Finland. Email: [email protected] Funding information Finnish Cardiovascular Foundation; Instrumentariumin Tiedesäätiö; FP7 Health, Grant/Award Numbers: F2‐2013‐602222, 2012‐3057392 Abstract 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. © 2018 The Authors. Journal of Cellular Physiology Published by Wiley Periodicals, Inc. J Cell Physiol. 2019;234:3744–3761.3744 | wileyonlinelibrary.com/journal/jcp --------------------------------------------------------------------------------------------------------------------------- This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Abbreviations: APOA1, apolipoprotein A‐I gene; APOB, apolipoprotein B gene; ASGR, asialoglycoprotein receptor; BMP4, bone morphogenic protein 4; CE, cholesteryl ester; Cer, ceramide; DAG, diacylglycerol; DE, definitive endoderm; EGF, epidermal growth factor; ELOVL, fatty acid elongase; FA, fatty acid; FADS, fatty acid desaturase; FASN, fatty acid synthase; Gb3, globotriaosylceramide; GCK, Glucokinase; Glc/GalCer, glucosyl/galactosylceramide; GSL, glycosphingolipid; HGF, hepatocyte growth factor; hiPSC, human induced pluripotent stem cell; HLC, hepatocyte‐like cell; hLTR, Human Liver Total RNA; LacCer, lactosylceramide; LPC, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; LPI, lysophosphatidylinositol; LPL, lysophospholipid; M1, Method 1; M2, Method 2; M3, Method 3; M4, Method 4; M5, Method 5; MUFA, monounsaturated fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PHH, primary human hepatocyte; PI, phosphatidylinositol; PL, phospholipid; PUFA, polyunsaturated fatty acid; SFA, saturated fatty acid; SL, sphingolipid; SM, sphingomyelin; TAG, triacylglycerol; UGCG, UDP‐glucose ceramide glucosyltransferase. KEYWORDS fatty acid (FA), gas chromatography, hepatocyte‐like cell (HLC), HepG2, human‐induced pluripotent stem cell (hiPSC), lipidomics, mass spectrometry (MS), primary human hepatocyte (PHH) 1 | INTRODUCTION The liver plays an important role in the regulation of many physiological functions of the body, including lipid and carbohydrate metabolism, glycolytic/urea metabolism, plasma protein synthesis, and the detoxification of a wide variety of molecules (Si‐Tayeb et al., 2010). Hepatocytes, which comprise about 70% of the liver’s mass, originate from the anterior portion of the definitive endoderm (DE), one of the three embryonic layers (Blouin, Bolender, & Weibel, 1977). Hepatocytes handle many crucial metabolic functions of the liver, including the synthesis of lipoproteins, triacylglycerols (TAGs), cholesterol, and phospholipids (PLs; Gordillo, Evans, & Gouon‐Evans, 2015). Primary human hepatocytes (PHHs) are currently considered the “gold standard”in cell modeling for studying, for example, liver physiology, toxicity, and lipid homeostasis. Typically, PHHs are obtained from cadaveric donors, but they are scarce and functionally heterogeneous, and it is hard to maintain them in a culture. When cultured, PHHs loose functionality relatively fast, and their liver‐ specific features progressively deteriorate, which particularly hampers long‐term studies (Elaut et al., 2006; Godoy et al., 2013). To address these limitations, various human hepatoma cell lines, including HepG2 and Huh7, have been used due to their ease of handling, unlimited life span, and stable phenotype. Nevertheless, they do not faithfully mirror the metabolic activities of healthy liver cells. In fact, the expression levels and profiles of genes involved in liver‐specific functions are poorly presented in these systems (Olsavsky et al., 2007). As a result, hepatoma cell lines have failed to predict the numerous adverse hepatotoxic side effects of new drugs (Castell, Jover, Martínez‐Jiménez, & Gómez‐Lechón, 2006). Alternatively, animal models, such as rats and mice, or animal primary hepatocytes have been widely used to study lipid metabolism and lipoprotein production in the liver (Kvilekval, Lin, Cheng, & Abumrad, 1994). However, the information gained from murine cells is not fully translatable to humans, and there are significant differences in lipid metabolism between the species. Animal models are also expensive and unsuitable for large‐scale screening. Furthermore, due to growing ethical concerns, there is an urgent need to reduce the use of rodents and other animal models in research. Human‐induced pluripotent stem cells (hiPSCs) provide an unlimited supply of tissue‐specific differentiated cell types for disease modeling and cell therapy. Hepatocytes differentiated from pluripotent stem cells circumvent the problem of the limited availability of cells faced when working with PHHs. They show very similar characteristics to PHHs; for instance, they secrete albumin as well as urea, and to some extent express drug transporters and cytochrome P450 enzymes (Kia et al., 2012). Unlike rodent hepatocytes and some human hepatoma cells, these hepatocyte‐like cells (HLCs) are sensitive to hepatitis C virus infection and support viral replication similar to PHHs (Schwartz et al., 2012). Patient‐specific iPSC lines provide us with an advantageous system of HLCs from patients of different genetic backgrounds, a prerequisite for the development of personalised medicine. The generation of hiPSC‐derived HLCs (hiPSC‐HLCs) in large quantities enables their use in modeling inborn errors of hepatic metabolism, understanding the molecular basis of liver cell differentiation, studying disease mechanisms, and facilitating drug discovery and safety. HLCs have already proven to be of great value in developing novel therapeutics (Medine et al., 2013; Szkolnicka et al., 2014) and identifying the noncoding micro‐RNAs regulating human liver damage (Szkolnicka et al., 2016; Yang et al., 2016). HLCs have also been successfully used as in vitro cell culture systems, for example, to recapitulate the pathophysiology of familial hypercholesterolemia (Cayo et al., 2012) and human cholesterol homeostasis (Krueger et al., 2013). Lipid defects are central to the pathogenesis of many common diseases, such as atherosclerosis (Meikle et al., 2011; Stübiger et al., 2012) and nonalcoholic fatty liver disease (Ruhanen et al., 2017; Younossi et al., 2016). Therefore, hiPSC‐HLCs offer a great platform for investigating the basic mechanisms of lipid metabolism and its dysregulation in a patient‐specific manner. However, to date, no detailed studies have yet been performed on the lipid profile and fatty acid (FA) metabolism of HLCs. To better use HLCs as a cell model to study the role of molecular lipids in liver diseases, it is essential to know their lipid profile in relation to actual human adult liver tissue and to the currently used cell models, namely PHHs and hepatoma cell lines. Hepatic differentiation methods have greatly improved over the last decade, enabling the efficient generation of high quality HLCs from pluripotent stem cells (Cameron et al., 2015; Hannan, Segeritz, Touboul, & Vallier, 2013; Hay et al., 2008; Kajiwara et al., 2012; Si‐Tayeb et al., 2010). Nevertheless, the experimental details and some differentiation factors differ between the current protocols, which might affect the phenotype of the HLCs. To reproduce the physiological conditions of the human liver, it is crucial to generate functional HLCs that are as similar as possible to PHHs. In this study, we describe five protocols for generating HLCs from hiPSCs and comprehensively compare the morphology, genetics, biochemistry, and functional traits of the HLCs produced. In addition, we compare the HLCs with the two most common cell models currently used—PHHs and HepG2 cells—as well as human liver tissue. Most importantly, the lipid profiles of HLCs,PHHs,andHepG2cellsareanalyzedbybothmass spectrometry (MS) and gas chromatography, and the similarities and main differences between the cell types are discussed in light of selected key genes involved in FA metabolism. Finally, the lipid profile of HLCs is fully characterized, and the potential and KIAMEHR ET AL. | 3745 limitations of HLCs as a model for studying lipid metabolism are evaluated. 2 | MATERIAL AND METHODS 2.1 | Ethical issues The study and patient recruitment have been approved by the Ethics Committee of Tampere University Hospital (approval number: R12123). All participants providing skin biopsies were adults more than 18 years old who had signed an informed consent form after receiving both oral and written descriptions of the study. 2.2 | hiPSC reprogramming and cell culture Three hiPSC lines (UTA.10100.EURCAs, UTA.11104.EURCAs, and UTA.11304.EURCCs) were generated directly from the fibroblasts of three individuals. Pluripotency was induced with the Sendai reprogramming kit (OCT4, SOX2, KLF4, C‐MYC; CytoTune; Life Technologies, USA) based on the protocol described by Ohnuki, Takahashi, Yamanaka, (2009) and Takahashi & Yamanaka, (2006). hiPSCs were then maintained as described before (Kiamehr et al., 2017). Details of the hiPSC cell culture are also described in the Supporting Information. 2.3 | Hepatic differentiation In all methods except Method 4 (M4), hiPSCs were transferred from mouse embryonic fibroblasts to Geltrex TM (USA; Cat: A14133‐ 01, 1:50 dilution), kept in mTeSR1 TM medium, and adapted to the changed culture conditions for a few passages before commencing the differentiation. In M4, hiPSCs were adapted on Laminin 521 (BioLamina, Lot: 80104) first, and the differentiation was completed on Laminin mix 111/521‐coated plates (3:1 ratio, 10 µg/ml). The hiPSCs used in all methods were at passage 20 or higher before commencing the differentiation. Three lines were differentiated using each method, except Method 5 (M5), which was applied to two cell lines. Figure 1 shows a schematic view of all five methods used in the study. Details of the five differentiation methods are described in detail in the Supporting Information. 2.4 | PHHs and HepG2 cells Cryopreserved PHHs (Cat. No. HMCPIS, Lot. HU8210, USA) were purchased from Gibco ® , and hepatocellular carcinoma cells (HepG2, ATCC‐HB‐8065, Lot. No. 59947519) were purchased from ATCC ™ . Both cells were plated according to the manufacturer’s instructions. PHHs were cultured in William’s E medium (A1217601, Gibco, USA) supplemented with cocktail B (Gibco, CM 4000) and dexamethasone, whereas the HepG2 cells were cultured in Dulbecco’s modified Eagle medium supplemented with 10% FBS. 2.5 | RT‐polymerase chain reaction RNA extraction and polymerase chain reaction (PCR) for pluripotency markers (OCT4,NANOG,SOX2, and SSEA4) were performed as published before (Manzini, Viiri, Marttila, & Aalto‐Setälä, 2015). 2.6 | Quantitative PCR (qPCR) analysis RNA samples were collected at the hiPSC, DE, and HLC stages, and RNA was extracted using an RNeasy kit (Qiagen, Germany, Cat. No. 74106). Complementary DNA (cDNA) was generated using a high capacity cDNA Reverse Transcription kit (Applied Biosystems) according to the manufacturer’s instructions in the presence of an RNase inhibitor. cDNA was multiplied either by the Power SYBR Green PCR Master Mix (Life Technology, Cat. No. 1408470, Austin, TX) and gene‐specific primers (OCT4,SOX17,FOXA2,AFP,ALB)orby the TaqMan Universal Master Mix (Applied Biosystems, 4304437, Austin, TX) and gene‐specific TaqMan probes (APOA1,APOB,FADS1, FADS2,ELOVL2,ELOVL5,FASN) using the BioRad CFX384 Real‐Time PCR Detection System. Values were normalized to GAPDH, which was used as an endogenous control, and relative quantification was FIGURE 1 A schematic representation of the five hepatic differentiation protocols used for generating hepatocyte‐like cells (HLCs). Differentiation in Method 1 and Method 2 (M1 and M2) were started with hiPSC colonies, in Method 3 and Method 4 (M3 and M4) with dissociated iPSC single cells, and in Method 5 (M5) with 24 hrs post‐cultured hiPSCs. Plates in Method 4 (M4) were coated with a mix of Laminin 111/521 (3:1 ratio) instead of the Geltrex TM used in the remainder of the methods. hiPSC: human‐induced pluripotent stem cell [Color figure can be viewed at wileyonlinelibrary.com] 3746 | KIAMEHR ET AL. calculated by the ΔΔCT method (Livak & Schmittgen, 2001). PHH was used as the reference sample. First Choice ® Human Liver Total RNA (hLTR, Cat. No. AM7960), purchased from Ambion®, was used as an extra control. The results from qPCR were compared for the HLCs and the three reference samples (PHHs, hLTR, and HepG2 cells). 2.7 | Immunostaining Cells were fixed, stained, and visualized as described before (Kiamehr et al., 2017). Details are also provided in the Supporting Information. The percentage of ALB‐positive cells and binuclear HLCs were calculated manually by counting HLCs in 3–5 stained areas and calculating the average. 2.8 | low‐density lipoprotein uptake The ability of the cells to uptake low‐density lipoprotein (LDL) was evaluated by incubating the HLCs with labeled LDL (Cell‐based assay kit, Cayman, USA, Cat. No. 10011125) for 4 hr, after which the cells were imaged by fluorescent microscopy. 2.9 | FACS analysis To analyze the number of CXCR4‐positive cells, endodermal cells were detached with Versene (Gibco ® , UK), suspended in 3%–5% bovine serum albumin buffer, stained with a PE‐conjugated CXCR4 antibody (R&D Systems FAB173P, Minneapolis, 10 µl for 10 6 cells) for 15 min at RT, washed three times, and analyzed using the Accuri TM C6 device (BD Biosciences). 2.10 | Albumin, urea, and TAG secretion At the late stage of hepatic differentiation, HLCs were evaluated for their functionality. The albumin, urea, and TAG content of the conditioned medium were determined, respectively, with the Human Albumin ELISA Quantitation kit (Bethyl Laboratory), the QuantiChrom TM Urea Assay Kit (BioAssay Systems, USA), and the Triglyceride Quantification Kit (BioVision Inc., Cat. No. K622‐100, USA) according to the manufacturers’instructions. The values were normalized to cell numbers. The results were then compared with the data from the PHHs and HepG2 cells, which were cultured in parallel with the HLCs. 2.11 | Lipid mass spectrometry 2.11.1 |Lipid sample preparation and extraction Lipids (sphingolipids [SL], cholesterol, glycerolipids, and glycerophospholipids) were extracted from the HLCs by Hamilton Robotics AB and studied by shotgun lipidomics. The detailed procedure is described by Kiamehr et al., (2017). 2.11.2 |Mass spectrometric analyses and data processing In the shotgun lipidomics (cholesteryl ester [CE], diacylglycerol [DAG], sphingomyelin [SM], lysophosphatidylcholine [LPC], lysophosphatidylethanolamine [LPE], lysophosphatidylserine, lysophosphatidylglycerol, and lysophosphatidylinositol [LPI]), lipid extracts were analyzed on a hybrid triple quadrupole/linear ion trap mass spectrometer (QTRAP 5500) equipped with a robotic nanoflow ion source (NanoMate, Advion Biosciences Inc., Ithaca, NJ) as described by Heiskanen, Suoniemi, Ta, Tarasov, & Ekroos, (2013). Molecular lipids were analyzed in positive ion mode using lipid class‐specific precursor ion or neutral loss scans (Ekroos, Chernushevich, Simons, & Shevchenko, 2002; Ekroos et al., 2003). Sphingolipids (ceramide [Cer], glucosyl/galactosylceramide [Glc/ GalCer], lactosylceramide [LacCer], and globotriaosylceramide [Gb3]) and molecular PLs (phosphatidylcholines [PC], phosphatidylethanolamines [PE], and phosphatidylinositols [PI]) were analyzed with a targeted approach using ultra‐high‐pressure liquid chromatography‐ mass spectrometry (UHPLC‐MS; Merrill, Sullards, Allegood, Kelly, & Wang, 2005). An analytical Acquity BEH C18, 2.1 × 50 mm column with a particle size of 1.7 µm (Waters, Milford, MA) heated to 60°C was used. Mobile phases consisted of 10 mM ammonium acetate in water with 0.1% formic acid (solvent A) and 10 mM ammonium acetate in acetonitrile:isopropanol (4:3, v/v) containing 0.1% formic acid (solvent B). The flow rate was set to 500 µl/min. Sphingolipids were separated with a 15 min linear gradient from 75% B to 100% B, while molecular PLs were analyzed using a 10 min gradient from 75% B to 80% B. Both sphingolipids and PLs were analyzed on a hybrid triple quadrupole/linear ion trap mass spectrometer (5500 QTRAP) equipped with an UHPLC system (CTC HTC PAL autosampler and Rheos Allegro pump or Shimadzu Nexera X2) using a multiple reaction monitoring based method in positive ion mode for sphingolipids and negative ion mode for molecular PLs. Curtain gas was set at 25; the ion spray voltage was set at 5000 V in positive ion mode and −4500 V in negative ion mode, and the ion source was heated to 400°C in positive mode and to 300°C in negative ion mode. The collision energy was optimized for each lipid class. Identified lipids were quantified by normalizing against their respective internal standard (Ejsing et al., 2006) and total protein concentrations in the cell sample. Total protein concentrations were determined using the Micro BCA ™ Protein Assay Kit (Thermo Scientific Pierce Protein Research Products) according to the manufacturer’s instructions. Data processing was performed by MultiQuant, LipidView (AB Sciex) software and SAS. 2.12 | FA gas chromatography To confirm our observations from the MS analysis of molecular lipids and to investigate the effect of the medium on the FA profile of the cells, the FA composition of HLCs, PHHs, and HepG2 cells and their media was analyzed by gas chromatography as described in detail by Kiamehr et al., (2017). Briefly, the acyl chains in the cell pellet KIAMEHR ET AL. | 3747 lipids or the lipid residues of the nitrogen‐dried media were converted to FA methyl esters (FAMEs) in a transesterification reaction with 1% methanolic H 2 SO 4 . The quantitative analysis of the FAMEs, which were extracted into hexane, was performed using a Shimadzu GC‐2010 Plus gas chromatograph with a flame‐ionization detector, and the FAME structures were identified by Shimadzu GCMSQP2010 Ultra with a mass selective detector. In both systems, the components of the FAME mixtures were separated in ZB‐wax capillary columns (30 m, 0.25 mm ID, 0.25 μm film; Phenomenex). The calculations of the FA compositions and concentrations followed standard procedures (Kiamehr et al., 2017), and the FAs were marked by using the abbreviations: [carbon number]:[number of double bonds] n‐[position of the first double bond calculated from the methyl end] (e.g., 22:6n‐3). 2.13 | Statistical analysis GraphPad Prism version 5.02 software was used for the data analysis. Data are presented as means ± standard deviation with n representing the number of independent experiments. The results were compared using one‐way analysis of variance, followed by Bonferroni’s multiple‐comparison test. A pvalue < 0.05 was considered statistically significant. 3 | RESULTS 3.1 | Cell morphology during hepatic differentiation In all methods, dramatic morphological changes were observed, particularly during the first few days of DE differentiation. In all methods except M5, migrating DE cells possessed a spiky morphology, whereas in M5, migrating cells were instead more round or square‐shaped (Supporting Information Figure S1a). In addition, the amount of cell death in M5 was considerably lower than in the other methods. No morphological differences were observed between the DE cells treated with CHIR 99021 (Method 1, M1) or Wnt3 (Methods 2–4, M2–4). Initiating differentiation with single cells in M3 and M4 did not yield a higher efficiency of DE formation compared with M1 and M2, which were started with colonies (Supporting Information Figures S1a and S3). However, we did observe cells with a DE morphology appearing one or even two days earlier in methods initiated with single cells compared with methods initiated with colonies (Supporting Information Figure S1a). At the hepatic specification stage, cells treated with basic fibroblast growth factor, bone morphogenic protein 4 (BMP4), and hepatocyte growth factor (HGF; M1) clearly had a different morphology compared with the cells treated with DMSO (M2–5; Supporting Information Figure S1b), which might imply different pathways toward hepatoblasts in those protocols. Binucleation is a feature of adult hepatocytes and generally considered a sign of terminal differentiation (Miyaoka & Miyajima, 2013). We found 29% of the PHHs and on average 10% of the HLCs to be binuclear ( Figures 1b and 2a). The HLCs differentiated by M4 showed the closest binuclearity (16%) to the PHHs. No individual cell line was shown to be more potent in generating binuclear cells. 3.2 | Characterization of DE and HLCs at the protein level hiPSCs expressed OCT4 protein, which was lost during the DE stage, while the expression of DE markers SOX17 and FOXA2 was upregulated (Supporting Information Figure S2a). The efficiency of the DE differentiation, estimated by measuring the CXCR4 expression by flow cytometry, did not differ across M1, M2, and M3 (Supporting Information Figure S2b). In M4 and M5, the amount of CXCR4‐positive cells was lower than in the other methods. The immature hepatic marker AFP was expressed in hepatic progenitor cells and remained expressed until the later stages in all five methods (Figure 2a). In addition, ALB, LDL receptor (LDL‐R), and asialoglycoprotein receptor (ASGR) were all expressed in mature HLCs (Figure 2a and Supporting Information Figure S5). The average of the ALB‐positive cells in M1 to M5 was 9.8%, 16.6%, 20.8%, 37.7%, and 31.5%, respectively, and the percentage in M4 was significantly higher than in M1, M2, and M3, but not in M5 (Figure 2b). More than 90% of M2‐to M5‐HLCs were positive for ASGR (data not shown). 3.3 | Gene regulation As expected, OCT4 was highly expressed at the iPSC stage, whereas at the DE stage, OCT4 was dramatically downregulated in most of the cell lines, and SOX17 and FOXA2 were highly expressed (Supporting Infomation Figure S6). Further differentiation toward HLCs resulted in significant downregulation of SOX17, whereas FOXA2 remained upregulated during the rest of the differentiation and maturation of the HLCs (Figure 2c and Supporting Infomation Figure S7). The HLCs expressed SOX17 at the same levels as hLTR. The level of FOXA2 in the HLCs was comparable to those in the reference samples PHH, hLTR, and HepG2. AFP was upregulated during the early and late hepatic differentiation stages, indicating the immature characteristic of the HLCs. The expression of AFP in the M2‐HLCs was statistically significantly higher than in the M1‐HLCs. ALB was dramatically upregulated in mature HLCs, up to 2 × 10 5 ‐fold compared with that in the hiPSCs, and its levels remained close but below those found in the PHHs. The expression of ALB in the M5‐HLCs was significantly higher than in the M1‐HLCs (p< 0.01) and M3‐HLCs (p< 0.05). The levels of ALB expression were comparable between the HepG2 and the PHHs; however, ALB expression was about 19‐fold higher in the hLTR when compared with the PHHs. 3.4 | Hepatic maturation and functionality The liver is responsible for producing serum albumin. Therefore, we evaluated the ability of HLCs to secrete albumin into the conditioned medium. All HLCs were able to secrete albumin (Figure 2d). Albumin secretion by UTA.11304 differentiated by M3 was about fourfold 3748 | KIAMEHR ET AL. FIGURE 2 Continued. KIAMEHR ET AL. | 3749 higher compared with the same cell line differentiated by M1 or M2, and twofold higher compared with M4 (data not shown), but no other statistically significant differences were observed between the differentiation protocols. However, PHHs secreted significantly larger amounts of albumin than the HLCs or even the HepG2 cells. The level of secreted urea by the cell lines differentiated by M3 and M4 was considerably higher compared with same lines differentiated by M1, M2, and M5 and relatively closer to the amount of urea secreted by the PHHs (Figure 2d). In fact, the level of urea secreted by the HLCs differentiated by M1, M2, and M5 was comparable to the HepG2 cells, which secreted 6.5‐fold less urea than the PHHs. TAG secretion in the cell lines differentiated by M3, M4, and M5 was at similar levels as in the PHHs (Figure 2d). However, cell lines differentiated by M1 and M2 secreted, on average, 5.9‐and 3.8‐fold more TAG than the PHHs. We were not able to detect secreted TAG in the HepG2 culture medium due to medium interference. Apolipoprotein A‐I(APOA1) encodes for apoA, which is the main protein component of high‐density lipoproteins (HDL). The product of apolipoprotein B (APOB) is the main protein component of very‐low‐density lipoprotein (VLDL) and LDL. The expression of both APOA1 and APOB in the HLCs was comparable to the HepG2 cells, PHHs, and hLTR, which further indicated the functionality of the HLCs (Figure 2c). Interestingly, both APOA1 and APOB were expressed almost 5.5‐fold more in hLTR compared with the PHHs. All the cell lines were able to uptake the labeled LDL from the culture medium (Supporting Information Figure S5). This was confirmed by staining the LDL‐R by monoclonal antibody (Supporting Information Figure S4). 3.5 | Lipid profiles of HLCs differentiated by different methods The HLCs differentiated by M3, M4, and M5 showed superior functionality as for TAG and urea secretion when compared with M1 and M2. In addition, the M5‐HLCs expressed higher ALB compared with the HLCs differentiated by M1 and M3. Therefore, we selected the HLCs differentiated by M3, M4, and M5, analyzed their lipid profile by MS, and compared their lipid profile. In addition, the lipid contents of their unconditioned media were studied to investigate the influence of culture media lipids and their FAs on the cells. Overall, 15 major lipid classes—including CE, DAG, PC, LPC, PI, LPI, PE, LPE, SM, Cer, LacCer, Glc/GalCer, and Gb3—were investigated, and altogether more than 150 molecular species were detected and studied (Supporting Information Tables S2 and S3). The lipid profile of the HLCs differentiated by M3, M4, and M5 closely resembled each other ( Figures ). In fact, only the levels of three PC species (PC 16:1–20:4, PC 17:0–18:1, and PC 17:0–20:4) were slightly, but statistically significantly, lower in M4 (Figure 3, marked by blue arrows) compared with the other methods. No other significant differences were found in any of the molecular species between the methods. When comparing the total levels of lipid classes, only Cer was found at significantly lower concentrations in the cells produced by M5 compared with those produced by M4 (Figure 5b). However, at the molecular species level, none of the Cer species differed significantly between M4 and M5 (Figure 5a and Supporting Information Table S2). 3.6 | Lipid profiles of HLCs, PHHs, and HepG2 cells Next, we compare the lipidomes of the HLCs with the PHHs and HepG2 cells and describe our findings for each lipid class separately. In addition, we highlight the differences and similarities between the PHHs and HepG2 cells. 3.6.1 |Phospholipids In the HLCs, the molecular species of PC, PE, and PI containing saturated FAs (SFAs) and monounsaturated FAs (MUFAs) were mostly similar to those in the PHHs, with the exceptions of PC 18:1/ 18:1 (p< 0.05), PC 17:0–18:1 (p< 0.001), and PE 18:0–18:1 (p< 0.05), which were statistically significantly higher in the HLCs (Figure 3, marked by red arrows). When the PHHs were compared with the HepG2 cells, a large number of species showed statistically significant differences, particularly the species containing 14:0, 16:0, and 18:1 FAs. In PC and PE, the molecular species containing polyunsaturated FAs (PUFAs) were present in significantly higher concentrations in the PHHs compared with both the HLCs and HepG2 cells. This difference was especially pronounced for the species containing an SFA coupled with FA 18:2 or its derivative 20:4 (e.g., 16:0–18:2, 18:0–18:2, 16:0–18:2, and 18:0–20:4). The HepG2 cells, however, contained more of the species where an MUFA was FIGURE 2 Characterization and functionality of hiPSC‐derived hepatocyte‐like cells (hiPSC‐HLCs) differentiated from three cell lines by five methods and their comparison to primary human hepatocytes (PHHs), HepG2 cells, and human liver total RNA (hLTR). (a) Immunostaining of cells for AFP (red) and ALB (green). Nuclei are stained with DAPI (blue). The red inset on the lower right shows the comparison of the morphology and binuclearity (red arrows) of M5‐HLCs and PHHs. The scale bar represents 200 µm for the HLCs and 100 µm for the PHHs and HepG2 cells. (b) The graph on the left shows the average percentage of binuclear HLCs in each method and their comparison to PHHs. The graph on the right shows the average percentage of ALB‐positive HLCs in each method. Each bar represents the mean ± SD of the manual counts from the immunostaining image analysis of at least five areas. (c) Real‐time qPCR analysis of the SOX17,FOXA2,AFP,ALB,APOA1, and APOB genes at the hiPSC and hepatic stage and their comparison to the reference samples. Each sample was run in triplicate and the bars represent the mean ± SD of three biological replicates from three individual cell lines. The gene expression data were normalized to the housekeeping gene GAPDH and are presented relative to the PHHs. (d) Biochemical analysis of the conditioned media from the HLCs for albumin, urea, and triacylglycerol. Values are normalized as 1 million cells per 24 hr. The bars represent the mean ± SD of three biological replicates of the three cell lines. *p< 0.05, **p< 0.01, ***p< 0.001. DAPI: 6‐diamidino‐2‐phenylindole; n.s.: not significant; qPCR: quantitative PCR; SD: standard deviation [Color figure can be viewed at wileyonlinelibrary.com] 3750 | KIAMEHR ET AL. coupled to a PUFA (or another MUFA). The total levels of PC and PI classes were similar among all three compared cell types. Total PE was, however, detected at lower levels in the HLCs compared with the PHHs (statistically significant in M4 and M5 vs the PHHs). This was mostly due to the higher levels of PE 18:0–18:2 and PE 18:0– 20:4 in the PHHs. The concentration of PC, PE, and PI in the HLC and PHH media was either zero or negligible, while the HepG2 medium contained high amounts of PC (12.9 µM) but only minimal amounts of PE and PI (Supporting Information Figure S7 and Table S3). The profile and concentration of molecular lysophospholipids (LPLs)— such as LPC, LPE, and LPI—in the HLCs were very close to those of the PHHs (Figure 3a). Consequently, the total LPL levels of the HLCs and PHHs were also similar (Figure 3b). The HepG2 cells, however, contained considerably higher levels of LPL as total levels and strikingly high levels of the molecular lyso‐species, with the 18:1 acyl residue in each LPL class (Figure 3 and Supporting Information Table S2). 3.6.2 |Neutral lipids CE concentration was the highest in theHepG2cells,intermediatein the HLCs, and the lowest in the PHHs. The CE species profile of the HLCs and HepG2 cells was different because 16 CE species showed statistically significantly higher concentrations in the HepG2 cells FIGURE 3 Lipidomic analysis of phospholipids (PLs) and lysophospholipids (LPLs) in the HLCs differentiated by M3, M4, and M5, and their comparison to PHH and HepG2 cell lipids. (a) Protein‐normalized concentration of molecular species detected in each class of PLs (Phosphatidylcholine [PC], phosphatidylinositol [PI], and phosphatidylethanolamine [PE]) as well as LPLs (lysophosphatidylcholine [LPC], lysophosphatidylinositol [LPI], and lysophosphatidylethanolamine [LPE]). The arrows refer to species that were found to be statistically significantly different between HLCs in M3, M4, and M5 (blue arrows) or between the HLCs and PHHs (red arrows) by one‐way analysis of variance. (b) Total concentrations of PLs and LPLs calculated from the sum of all the molecular species in those specific classes. Each sample was run in triplicate and the bars represent the mean ± standard deviation of the studied cell lines [Color figure can be viewed at wileyonlinelibrary.com] KIAMEHR ET AL. | 3751 (Figure 4). When interpreting these differences, the effect of medium CE concentration was considered. The HepG2 medium contained 154 µM of CE, whereas the CE concentrations were negligible in the HLC and PHH media (Supporting Information Figure S7 and Table S3). Similar to PLs, the HLCs contained higher levels of DAG species with FA 18:1 when compared with the PHHs. However, the level of major DAG species 16:0–18:1 detected in the HLCs was clearly closer to that detected in the PHHs compared with what was found in the HepG2 cells. The PHHs, on the other hand, contained higher levels of DAG species with FA 18:2 (the minor species DAG 18: 1–18:2 being an exception with its equally low levels in the HLCs, PHHs, and HepG2 cells). As also observed in PC, the DAG species with relatively short chain FAs (e.g., 14:0, 16:0, and 16:1) were detected at higher concentrations in the HepG2 cells than in the HLCs and PHHs (Figure 4A). 3.6.3 |Sphingolipids Despite higher total SM concentration, HLCs mimicked the SM profile of PHHs and HepG2 cells, except that SM d18:1/15:0 and SM d18:1/16:1 were present at statistically significantly higher concentrations in the HepG2 cells (Figure 5a). Both the HLCs and HepG2 cells contained significantly higher levels of SM d18:1/16:0 compared with the PHHs. The level of SM in the HLC and PHH media was undetectable, whereas the HepG2 medium contained 6.5 µM of SM (Supporting Information Figure S7 and Table S3). In terms of the overall SL profile, the HepG2 cells situated between the PHHs and HLCs. In fact, lipid class data showed that the HLCs contained lower levels of Cer but higher levels of LacCer, Glc/ GalCer, and Gb3 (members of glycosphingolipid [GSL] family) compared with the PHHs (Figure 5b). Closer examination showed that Cers and, particularly, the saturated species with long‐and very‐ long‐chain FAs (e.g., Cer d18:0/22:0 and Cer d18:1/24:0) were higher in the PHHs (Figure 5a), in fact, the Cer species profile of the HLCs resembled more that of the HepG2 cells. On the other hand, the HLCs contained more GSLs—especially the species d18:1/16:0, d18:1/24:0, and d18:1/24:1—and the differences were the most pronounced in the Glc/GalCer class. The HepG2 medium contained trace amounts of SLs, and the concentration of SLs in the HLC and PHH media was either negligible or undetectable (Supporting Information Figure S7 and Table S3). 3.7 | FA analysis The HLCs contained 15 mol% PUFAs versus 29 mol% and 10.5 mol % in the PHHs and HepG2 cells, respectively (Figure 6a). The FA FIGURE 4 Lipidomic analysis of cholesteryl ester (CE) and diacylglycerol (DAG) in the HLCs differentiated by M3, M4, and M5, and their comparison to PHH and HepG2 cell lipids. (a) Protein‐normalized concentration of molecular species detected for the CE and DAG lipid class. (b) Total concentrations of CE and DAG calculated from the sum of all the molecular species in those specific classes. Each sample was run in triplicate and the bars represent the mean ± standard deviation of the studied cell lines [Color figure can be viewed at wileyonlinelibrary.com] 3752 | KIAMEHR ET AL. The authors gratefully acknowledge the Tampere facility of iPS Cells and Flow Cytometry for their services. CONFLICTS OF INTEREST The authors declare no conflicts of interest in this study. ORCID Mostafa Kiamehr http://orcid.org/0000-0003-1894-3237 REFERENCES Anatskaya, O. V., & Vinogradov, A. E. (2007). 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How to cite this article: Kiamehr M, Alexanova A, Viiri LE, et al. hiPSC‐derived hepatocytes closely mimic the lipid profile of primary hepatocytes: A future personalised cell model for studying the lipid metabolism of the liver. J Cell Physiol. 2019;234:3744–3761. https://doi.org/10.1002/jcp.27131 KIAMEHR ET AL. | 3761