Flow cytometric quantification of neutral lipids in a human skin stem cell-derived model of NASH

Non-alcoholic steatohepatitis (NASH) is a severe chronic liver disease that affects 3 to 5 percent of the world population. It is characterized by hepatic lipid accumulation and inflammation and can progress towards fibrosis, cirrhosis and hepatocellular carcinoma. Until today, no drug has been approved for the treatment of NASH. This delay relates to the complex pathogenesis of NASH and also to a lack of appropriate predictive preclinical testing systems. Furthermore, the human specificity of the NASH pathology hampers a fortiori clinical translation of animal studies. Therefore, we recently employed human skin-derived precursors (hSKP) differentiated to hepatocyte-like cells (hSKP-HPC) as a human-relevant cell source for modelling NASH in vitro. Using this in vitro NASH model, it was possible to test novel drugs being developed for anti-NASH therapy, such as elafibranor. Since steatosis is an important aspect of NASH and multiple drugs are being developed to decelerate and reduce lipid accumulation in the liver, we optimized a flow cytometric method for quantifying neutral lipids in ‘NASH’-triggered hSKP-HPC. This methodology enables efficient identification of anti-steatotic properties of new medicines. • NASH-triggered hSKP-HPC robustly accumulate lipids intracellularly. • Flow cytometric quantification of neutral lipids in NASH-triggered hSKP-HPC allows for accurate determination of the steatotic response. • This method enables efficient identification of potential anti-steatotic drugs in a human-specific model


Specifications
Pharmacology, Toxicology and Pharmaceutical Science More specific subject area: Preclinical drug testing Method name: Flow cytometric quantification of neutral lipids in human skin-derived hepatic cells Name and reference of original method:

Method details
Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent chronic liver disease that is closely associated with the metabolic syndrome [1] . NAFLD encompasses a spectrum of diseases ranging from liver steatosis to severe non-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis and hepatocellular carcinoma. NASH is characterized by hepatic lipid accumulation and inflammation and fulfills a key role in NAFLD since it is considered as the tipping point to the latter life-threatening conditions [2] . At present, no drug has been approved to treat or cure NASH [3] . Liver steatosis is an important parameter of NASH [4 , 5] , hence, the majority of newly-developed anti-NASH drugs focuses, apart from tackling hepatic inflammation, also on liver steatosis. Animal models are traditionally used for preclinical NASH research. Yet, important interspecies differences exist between rodents and humans with regard to the development of NASH and investigation of anti-NASH therapies. For example, when peroxisome proliferator-activated receptor alpha (PPAR-α) agonists, which are drugs that are used for treating dyslipidemia, are administered to rodents, these animals develop liver cancer [6] . Considering that this complex disease could be solved using PPAR agonists highlights the need for human-relevant models in early anti-NASH drug development [3] .
Our group has developed a protocol for differentiating human skin-derived precursors (hSKPs) to hepatic cells (hSKP-HPC) by mimicking the liver embryogenesis in vitro . hSKP-HPC exhibit a mixed phenotype of mature ( ALB ) and immature ( EPCAM, NCAM2, PROM1 ) hepatocytes [7] . The applicability of hSKP-HPC has been earlier demonstrated for drug-induced liver injury (DILI) by using the reference compounds acetaminophen [7] , sodium valproate [8] and amiodarone [9] , respectively inducing acute liver injury, steatosis and phospholipidosis. hSKP-HPC have been also employed for in vitro modelling of metabolically-induced liver steatosis and NASH [10] . When hSKP-HPC were exposed to fatty acids, insulin, glucose and inflammatory cytokines, these cells massively accumulated lipids and secreted inflammatory cytokines characteristic to NASH. When these 'NASH'-triggered cells were challenged with an anti-NASH drug under development ( i.e. elafibranor, a PPAR-α/ δ agonist), clear anti-inflammatory and anti-steatotic responses could be observed, of which the latter was mediated by different cellular processes including fatty acid transport, endoplasmic reticulum stress and de novo lipogenesis. These successful applications are a consequence of the fact that hSKPs acquire a strong lipid metabolism upon hepatic differentiation to hSKP-HPC. Furthermore, hSKP-HPC recently showed to mimic the anti-steatotic responses induced by a series of PPAR agonists in a similar way as primary human hepatocytes, being the gold standard for hepatic in vitro modelling, emphasizing the position of hSKP-HPC for modelling NASH in vitro [11] . Considering the restricted availability and fragility of primary human hepatocytes, we optimized a flow cytometric method to quantify the antisteatotic response induced by potential anti-NASH pharmaceuticals in hSKP-HPC. This methodology can be implemented in early preclinical drug development for testing of anti-steatotic properties of new chemical entities (NCE) in a human-relevant way.

hSKP-HPC culture
hSKPs are isolated and differentiated as earlier reported [7 , 12] . Briefly, hSKPs are isolated from human (fore)skin dermis using a series of enzymatic and mechanical steps. hSKPs aggregate and form spheres through clonal expansion in a medium containing epidermal growth factor (EGF) and fibroblast growth factor 2 (FGF2). After 2-3 weeks, the spheres are dissociated into single cells and grown in adherent monolayer cultures. Cells cultured in monolayers can be further expanded and cryopreserved. hSKPs are differentiated on rat tail collagen type I-coated cell culture recipients and are required to reach 90-95% confluence before hepatic differentiation is initiated. After 24 days of hepatic differentiation, hSKP-HPCs are obtained. A graphical representation of the hSKP culture and hepatic differentiation to hSKP-HPC is given in Fig. 1 . For the purpose of this flow cytometric analysis, the cells can be cultured in either 6-or 24-multiwell (MW) plates. One well from a 6-MW plate or four wells from a 24-MW plate are sufficient per sample for performing the analysis.

Preparation of exposure solutions
hSKP-HPC control media are composed of the medium used for the final steps of the differentiation (D 24 medium) [7] supplemented with fatty acid free BSA (0.14% (m/v)) and DMSO (0.15% (v/v)).
hSKP-HPC 'NASH' media are composed of D 24 medium supplemented with glucose (Sigma-Aldrich) ((4.5 mg/mL) together with insulin (100 nM), sodium oleate (65 μM), IL-1 β (25 ng/mL), TGF-β (8 ng/mL) and TNF-α (50 ng/mL). Palmitic acid needs to be 37 °C pre-warmed and added quickly to 37 °C pre-warmed medium, directly followed by a 30 s. vortex step to avoid precipitation. Hereafter either the compound or its vehicle (DMSO) can be added (1:10 0 0). Note: in this protocol the compound is dissolved to obtain a 10 0 0-fold concentrated stock solution. Some compounds are less soluble, which implies that also higher DMSO concentrations need to be added to the control and vehicle media, however final DMSO concentration cannot exceed 0,5% (v/v). All solutions are filtered through a medium filter with 200 μM pore size before exposure to the cells.

Exposure of hSKP-HPC
Expose the cells to 50 0 μL or 150 0 μL media per well of the 24-or 6-MW plates, respectively. Incubate hSKP-HPC for 24 h at 37 °C in a humidified incubator with 5% CO 2 .

Harvesting of hSKP-HPC and dissociation into single cells
After 24 h of incubation, the media are gently removed from the cells and replaced by either 200 μL (24-MW plate format) or 500 μL (6-MW plate format) trypsin solution (TrypLE reagent, Thermo Fisher Scientific) per well. Then, the cells are incubated for 10 min. at 37 °C in a humidified incubator with 5% CO 2 . After 10 min., cell detachment is evaluated under a phase contrast microscope (Nikon Eclipse) at 10 x magnification. If the cells do not come loose after 10 min., the culture plate is gently tapped on the sides and incubated for 5 more min. Subsequently, 500 μL (24-MW format) or 1500 μL (6-MW format) 37 °C pre-warmed PBS is added to each well to stop the action of the TrypLE reagent. hSKP-HPC are resuspended into single cells by gentle pipetting and are collected in a 15 mL conical tube. The wells are rinsed with 37 °C pre-warmed PBS using the same volumes. One additional control sample is included for setting up the flow cytometer.

Sample preparation
The conical 15 mL tubes containing hSKP-HPC are centrifuged during 5 min at 470 g. After 5 min., the supernatant is gently aspirated using a 5 mL pipet and the pellet is resuspended with 1 mL PBS

Instrument settings
The Attune R Acoustic Focusing Cytometer (Life Technologies) is used for flow cytometric analysis. The flow cytometer is set at a flow rate of 500 μL per second with a maximum detection of 10 0.0 0 0 events. The specific fluorescence detection channels are chosen for BODIPY TM 493/503 (blue laser (BL) −1 channel) and Hoechst 33,342 (violet laser (VL) −1 channel). After quality control of the flow cytometer according to the manufacturer's instructions, the unstained control sample is ran for adjusting the voltages ( Fig. 2 A) of the forward scatter (FSC), side scatter (SSC), BL-1 and VL-1 channels. The voltages of FSC and SSC need to be adjusted so that the cell population spreads in the main area of the dot plot, and can be distinguished from the debris material ( Fig. 2 B). Fig. 2 C shows the unspecific signals generated on the VL-1 and BL-1 channels from an unstained sample. Voltages are ideally set to obtain unspecific relative fluorescence signals from maximal 10 ³ à 10 4 to allow for a wide enough measuring range (especially for the BL-1 channel to measure a shift in neutral lipid load). Hereafter, gates ('Hoechst', blue and 'Bodipy', yellow) are set in order to only measure signals derived from the specific dyes.

Data analysis
Debris, which can be observed as an accumulation of events having almost baseline forward-and side scatters, is excluded from the analysis ( Fig. 3 A). Hereafter, the BODIPY TM 493/503 neutral lipid dye signal is set as a function of Hoechst 33,342-positive cells ( Fig. 3 B). An induction in intensity of BODIPY TM 493/503 is observed in hSKP-HPC exposed to 'NASH' triggers compared to control hSKP-HPC ( Fig. 3 C). Exposure to elafibranor induces a shift to the left, indicating a decrease in intracellular lipids.
Quantification of this observation can be done by plotting the median fluorescence signals for each condition. As an example, Fig. 3 D shows a significant induction in BODIPY TM 493/503 intensity in hSKP-HPC triggered with 'NASH' compounds, which decreases upon increasing elafibranor concentrations. As such, this method enables the testing of anti-steatotic properties of potential anti-NASH compounds using a human-relevant in vitro model.

Declaration of Competing Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.