Affordable blood culture systems from China: in vitro evaluation for use in resource-limited settings.

BACKGROUND: Bloodstream infections (BSI) pose a significant threat due to high mortality rates and the challenges posed by antimicrobial resistance (AMR). In 2019, an estimated 4.95 million deaths were linked to bacterial AMR. The highest impact was seen in resource-limited settings (RLS). For diagnosis of BSI, performant continuously-monitoring blood culture systems (CMBCS) have been optimized. However, in RLS, the implementation of CMBCS is hindered by budget constraints and unsuitable environmental conditions. Manufacturers from growing economies are currently producing affordable in vitro diagnostics, which could fill the gap in capacity, but so far these are not established outside their domestic markets. METHODS: This study evaluated the performance, usability, and interchangeability of Chinese CMBCS in a laboratory setting using simulated blood cultures with a panel of 20 BSI-associated strains. Four systems were selected for the assessment: Autobio BC60, Mindray TDR60, Scenker Labstar50, and DL-biotech DL-60. FINDINGS: Overall, all evaluated CMBCS demonstrated good performance with high yield (96.7-100%) and specificity (97.5-100%), comparable to the reference system (bioMérieux 3D). In addition, when used as "manual" blood cultures in a conventional incubator with visual growth detection, performance was also satisfactory: yield was between 90 and 100% and specificity was 100% for all BCBs. Both the CMBCS and the BCBs were easy to use and lot-to-lot variability in BCBs was minimal. The interchangeability testing indicated that the BCBs from different brands (all except Scenker) were compatible with the various automates, further highlighting the potential for a harmonized "universal BCB." INTERPRETATION: Based on this in vitro study, we recommend the use of these systems in settings with challenging environments and limited resources. The Autobio system performed best for automatic detection and DL-Biotech BCBs for manual cultures respectively (combination of performance, price, usability). The appropriateness for use in RLS should still be confirmed in a field study. FUNDING: The study was funded by FIND.

Inactivation of Foodborne Pathogens by Lactiplantibacillus Strains during Meat Fermentation: Kinetics and Mathematical Modelling.

This study examined the effect of beef fermentation with Lactiplantibacillus paraplantarum (L) PTCC 1965, Lactiplantibacillus (L) plantarum subsp. plantarum PTCC 1745, and Lactiplantibacillus (L) pentosus PTCC 1872 bacteria on the growth of pathogenic bacteria, including Salmonella (S) Typhi PTCC 1609 and Staphylococcus (S) aureus PTCC 1826. The growth of lactic acid bacteria (LAB) and the effect of fermentation on pathogenic bacteria were studied using Weibull: biphasic linear and competitive models. The results showed that the rate of pH reduction was lower in the early stages and increased as the microbial population grew. The α parameter was lower for L. plantarum subsp. plantarum compared to L. paraplantarum and L. pentosus. The comparison of the α parameter for bacterial growth and pH data showed that the time interval required to initiate the rapid growth phase of the bacteria was much shorter than that for the rapid pH reduction phase. The pH value had a 50% greater effect on the inactivation of S. Typhi when compared to the samples containing L. plantarum subsp. plantarum and L. pentosus. The same parameter was reported to be 72% for the inactivation of St. aureus. In general, during the fermentation process, LAB strains caused a decrease in pH, and as a result, reduced the growth of pathogens, which improves consumer health and increases the food safety of fermented meat.

Fragility Score: a REMS-based indicator for the prediction of incident fragility fractures at 5 years.

BACKGROUND: Accurate estimation of the imminent fragility fracture risk currently represents a challenging task. The novel Fragility Score (FS) parameter, obtained during a Radiofrequency Echographic Multi Spectrometry (REMS) scan of lumbar or femoral regions, has been developed for the non-ionizing estimation of skeletal fragility. AIMS: The aim of this study was to assess the performance of FS in the early identification of patients at risk for incident fragility fractures with respect to bone mineral density (BMD) measurements. METHODS: Data from 1989 Caucasians of both genders were analysed and the incidence of fractures was assessed during a follow-up period up to 5 years. The diagnostic performance of FS to discriminate between patients with and without incident fragility fracture in comparison to that of the BMD T-scores measured by both Dual X-ray Absorptiometry (DXA) and REMS was assessed through ROC analysis. RESULTS: Concerning the prediction of generic osteoporotic fractures, FS provided AUC = 0.811 for women and AUC = 0.780 for men, which resulted in AUC = 0.715 and AUC = 0.758, respectively, when adjusted for age and body mass index (BMI). For the prediction of hip fractures, the corresponding values were AUC = 0.780 for women and AUC = 0.809 for men, which became AUC = 0.735 and AUC = 0.758, respectively, after age- and BMI-adjustment. Overall, FS showed the highest prediction ability for any considered fracture type in both genders, resulting always being significantly higher than either T-scores, whose AUC values were in the range 0.472-0.709. CONCLUSION: FS displayed a superior performance in fracture prediction, representing a valuable diagnostic tool to accurately detect a short-term fracture risk.

Evaluation of InTray Cassettes Directly from Blood Cultures for the Diagnosis of Sepsis in Clinical Bacteriology Laboratories as an Alternative to Classic Culture Media.

Culture media is fundamental in clinical bacteriology for the detection and isolation of bacterial pathogens. However, in-house media preparation could be challenging in low-resource settings. InTray® cassettes (Biomed Diagnostics) could be a valid alternative as they are compact, ready-to-use media preparations. In this study, we evaluate the use of two InTray media as a subculture alternative for the diagnosis of bloodstream infections: the InTray® Müller-Hinton (MH) chocolate and the InTray® Colorex™ Screen. The InTray MH chocolate was evaluated in 2 steps: firstly, using simulated positive blood cultures (reference evaluation study), and secondly, using positive blood cultures from a routine clinical laboratory (clinical evaluation study). The Colorex Screen was tested using simulated poly-microbial blood cultures. The sensitivity and specificity of the InTray MH chocolate were respectively 99.2% and 90% in the reference evaluation study and 97.1% and 88.2% in the clinical evaluation study. The time to detection (TTD) was ≤20 h in most positive blood cultures (99.8% and 97% in the two studies, respectively). The InTray® MH Chocolate agar showed good performance when used directly from clinical blood cultures for single bacterial infections. However, mixed flora is more challenging to interpret on this media than on Colorex™ Screen, even for an experienced microbiologist.

Validation of Three MicroScan® Antimicrobial Susceptibility Testing Plates Designed for Low-Resource Settings.

Easy and robust antimicrobial susceptibility testing (AST) methods are essential in clinical bacteriology laboratories (CBL) in low-resource settings (LRS). We evaluated the Beckman Coulter MicroScan lyophilized broth microdilution panel designed to support Médecins Sans Frontières (MSF) CBL activity in difficult settings, in particular with the Mini-Lab. We evaluated the custom-designed MSF MicroScan Gram-pos microplate (MICPOS1) for Staphylococcus and Enterococcus species, MSF MicroScan Gram-neg microplate (MICNEG1) for Gram-negative bacilli, and MSF MicroScan Fastidious microplate (MICFAST1) for Streptococci and Haemophilus species using 387 isolates from routine CBLs from LRS against the reference methods. Results showed that, for all selected antibiotics on the three panels, the proportion of the category agreement was above 90% and the proportion of major and very major errors was below 3%, as per ISO standards. The use of the Prompt inoculation system was found to increase the MIC and the major error rate for some antibiotics when testing Staphylococci. The readability of the manufacturer's user manual was considered challenging for low-skilled staff. The inoculations and readings of the panels were estimated as easy to use. In conclusion, the three MSF MicroScan MIC panels performed well against clinical isolates from LRS and provided a convenient, robust, and standardized AST method for use in CBL in LRS.

Biphasic versus monophasic manual blood culture bottles for low-resource settings: an in-vitro study.

BACKGROUND: Manual blood culture bottles (BCBs) are frequently used in low-resource settings. There are few BCB performance evaluations, especially evaluations comparing them with automated systems. We evaluated two manual BCBs (Bi-State BCB and BacT/ALERT BCB) and compared their yield and time to growth detection with those of automated BacT/ALERT system. METHODS: BCBs were spiked in triplicate with 177 clinical isolates representing pathogens common in low-resource settings (19 bacterial and one yeast species) in adult and paediatric volumes, resulting in 1056 spiked BCBs per BCB system. Growth in manual BCBs was evaluated daily by visually inspecting the broth, agar slant, and, for BacT/ALERT BCB, colour change of the growth indicator. The primary outcomes were BCB yield (proportion of spiked BCB showing growth) and time to detection (proportion of positive BCB with growth detected on day 1 of incubation). 95% CI for yield and growth on day 1 were calculated using bootstrap method for clustered data using. Secondary outcomes were time to colony for all BCBs (defined as number of days between incubation and colony growth sufficient to use for further testing) and difference between time to detection in broth and on agar slant for the Bi-State BCBs. FINDINGS: Overall yield was 95·9% (95% CI 93·9-98·0) for Bi-State BCB and 95·5% (93·3-97·8) for manual BacT/ALERT, versus 96·1% (94·0-98·1) for the automated BacT/ALERT system (p=0·61). Day 1 growth was present in 920 (90·8%) of 1013 positive Bi-State BCB and 757 (75·0%) of 1009 positive manual BacT/ALERT BCB, versus 1008 (99·3%) of 1015 automated bottles. On day 2, detection rates were 100% for BI-State BCB, 97·7% for manual BacT/ALERT BCB, and 100% for automated bottles. For Bi-State BCB, growth mostly occurred simultaneously in broth and slant (81·7%). Sufficient colony growth on the slant to perform further tests was present in only 44·1% of biphasic bottles on day 2 and 59·0% on day 3. INTERPRETATION: The yield of manual BCB was comparable with the automated system, suggesting that manual blood culture systems are an acceptable alternative to automated systems in low-resource settings. Bi-State BCB outperformed manual BacT/ALERT bottles, but the agar slant did not allow earlier detection nor earlier colony growth. Time to detection for manual blood culture systems still lags that of automated systems, and research into innovative and affordable methods of growth detection in manual BCBs is encouraged. FUNDING: Médecins Sans Frontières and Department of Economy, Science and Innovation of the Flemish Government.

Considerations in evaluating equipment-free blood culture bottles: A short protocol for use in low-resource settings.

Use of equipment-free, "manual" blood cultures is still widespread in low-resource settings, as requirements for implementation of automated systems are often not met. Quality of manual blood culture bottles currently on the market, however, is usually unknown. An acceptable quality in terms of yield and speed of growth can be ensured by evaluating the bottles using simulated blood cultures. In these experiments, bottles from different systems are inoculated in parallel with blood and a known quantity of bacteria. Based on literature review and personal experiences, we propose a short and practical protocol for an efficient evaluation of manual blood culture bottles, aimed at research or reference laboratories in low-resource settings. Recommendations include: (1) practical equivalence of horse blood and human blood; (2) a diverse selection of 10 to 20 micro-organisms to be tested (both slow- and fast-growing reference organisms); (3) evaluation of both adult and pediatric bottle formulations and blood volumes; (4) a minimum sample size of 120 bottles per bottle type; (5) a formal assessment of usability. Different testing scenarios for increasing levels of reliability are provided, along with practical tools such as worksheets and surveys that can be used by laboratories wishing to evaluate manual blood culture bottles.

Increasing the Microfabrication Performance of Synthetic Hydrogel Precursors through Molecular Design.

Implementation of hydrogel precursors in two-photon polymerization (2PP) technology provides promising opportunities in the tissue engineering field thanks to their soft characteristics and similarity to extracellular matrix. Most of the hydrogels, however, are prone to post-fabrication deformations, leading to a mismatch between the computer-aided design and the printed structure. In the present work, we have developed novel synthetic hydrogel precursors to overcome the limitations associated with 2PP processing of conventional hydrogel precursors such as post-processing deformations and a narrow processing window. The precursors are based on a poly(ethylene glycol) backbone containing urethane linkers and are, on average, functionalized with six acrylate terminal groups (three on each terminal group). As a benchmark material, we exploited a precursor with an identical backbone and urethane linkers, albeit functionalized with two acrylate groups, that were reported as state-of-the-art. An in-depth characterization of the hexafunctional precursors revealed a reduced swelling ratio (<0.7) and higher stiffness (>36 MPa Young's modulus) compared to their difunctional analogs. The superior physical properties of the newly developed hydrogels lead to 2PP-based fabrication of stable microstructures with excellent shape fidelity at laser scanning speeds up to at least 90 mm s-1, in contrast with the distorted structures of conventional difunctional precursors. The hydrogel films and microscaffolds revealed a good cell interactivity after functionalization of their surface with a gelatin methacrylamide-based coating. The proposed synthesis strategy provides a one-pot and scalable synthesis of hydrogel building blocks that can overcome the current limitations associated with 2PP fabrication of hydrogel microstructures.

AMR in low-resource settings: Médecins Sans Frontières bridges surveillance gaps by developing a turnkey solution, the Mini-Lab.

BACKGROUND: In low- and middle-income countries (LMICs), data related to antimicrobial resistance (AMR) are often inconsistently collected. Humanitarian, private and non-governmental medical organizations (NGOs), working with or in parallel to public medical systems, are sometimes present in these contexts. Yet, what is the role of NGOs in the fight against AMR, and how can they contribute to AMR data collection in contexts where reporting is scarce? How can context-adapted, high-quality clinical bacteriology be implemented in remote, challenging and underserved areas of the world? OBJECTIVES: The aim was to provide an overview of AMR data collection challenges in LMICs and describe one initiative, the Mini-Lab project developed by Médecins Sans Frontières (MSF), that attempts to partially address them. SOURCES: We conducted a literature review using PubMed and Google scholar databases to identify peer-reviewed research and grey literature from publicly available reports and websites. CONTENT: We address the necessity of and difficulties related to obtaining AMR data in LMICs, as well as the role that actors outside of public medical systems can play in the collection of this information. We then describe how the Mini-Lab can provide simplified bacteriological diagnosis and AMR surveillance in challenging settings. IMPLICATIONS: NGOs are responsible for a large amount of healthcare provision in some very low-resourced contexts. As a result, they also have a role in AMR control, including bacteriological diagnosis and the collection of AMR-related data. Actors outside the public medical system can actively contribute to implementing and adapting clinical bacteriology in LMICs and can help improve AMR surveillance and data collection.

Evaluation of MicroScan Bacterial Identification Panels for Low-Resource Settings.

Bacterial identification is challenging in low-resource settings (LRS). We evaluated the MicroScan identification panels (Beckman Coulter, Brea, CA, USA) as part of Médecins Sans Frontières' Mini-lab Project. The MicroScan Dried Overnight Positive ID Type 3 (PID3) panels for Gram-positive organisms and Dried Overnight Negative ID Type 2 (NID2) panels for Gram-negative organisms were assessed with 367 clinical isolates from LRS. Robustness was studied by inoculating Gram-negative species on the Gram-positive panel and vice versa. The ease of use of the panels and readability of the instructions for use (IFU) were evaluated. Of species represented in the MicroScan database, 94.6% (185/195) of Gram-negative and 85.9% (110/128) of Gram-positive isolates were correctly identified up to species level. Of species not represented in the database (e.g., Streptococcus suis and Bacillus spp.), 53.1% out of 49 isolates were incorrectly identified as non-related bacterial species. Testing of Gram-positive isolates on Gram-negative panels and vice versa (n = 144) resulted in incorrect identifications for 38.2% of tested isolates. The readability level of the IFU was considered too high for LRS. Inoculation of the panels was favorably evaluated, whereas the visual reading of the panels was considered error-prone. In conclusion, the accuracy of the MicroScan identification panels was excellent for Gram-negative species and good for Gram-positive species. Improvements in stability, robustness, and ease of use have been identified to assure adaptation to LRS constraints.

Human hepatic in vitro models reveal distinct anti-NASH potencies of PPAR agonists.

Non-alcoholic steatohepatitis (NASH) is a highly prevalent, chronic liver disease characterized by hepatic lipid accumulation, inflammation, and concomitant fibrosis. Up to date, no anti-NASH drugs have been approved. In this study, we reproduced key NASH characteristics in vitro by exposing primary human hepatocytes (PHH), human skin stem cell-derived hepatic cells (hSKP-HPC), HepaRG and HepG2 cell lines, as well as LX-2 cells to multiple factors that play a role in the onset of NASH. The obtained in vitro disease models showed intracellular lipid accumulation, secretion of inflammatory chemokines, induced ATP content, apoptosis, and increased pro-fibrotic gene expression. These cell systems were then used to evaluate the anti-NASH properties of eight peroxisome proliferator-activated receptor (PPAR) agonists (bezafibrate, elafibranor, fenofibrate, lanifibranor, pemafibrate, pioglitazone, rosiglitazone, and saroglitazar). PPAR agonists differently attenuated lipid accumulation, inflammatory chemokine secretion, and pro-fibrotic gene expression.Based on the obtained readouts, a scoring system was developed to grade the anti-NASH potencies. The in vitro scoring system, based on a battery of the most performant models, namely PHH, hSKP-HPC, and LX-2 cultures, showed that elafibranor, followed by saroglitazar and pioglitazone, induced the strongest anti-NASH effects. These data corroborate available clinical data and show the relevance of these in vitro models for the preclinical investigation of anti-NASH compounds.

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.

Anti-NASH Drug Development Hitches a Lift on PPAR Agonism.

Non-alcoholic fatty liver disease (NAFLD) affects one-third of the population worldwide, of which a substantial number of patients suffer from non-alcoholic steatohepatitis (NASH). NASH is a severe condition characterized by steatosis and concomitant liver inflammation and fibrosis, for which no drug is yet available. NAFLD is also generally conceived as the hepatic manifestation of the metabolic syndrome. Consequently, well-established drugs that are indicated for the treatment of type 2 diabetes and hyperlipidemia are thought to exert effects that alleviate the pathological features of NASH. One class of these drugs targets peroxisome proliferator-activated receptors (PPARs), which are nuclear receptors that play a regulatory role in lipid metabolism and inflammation. Therefore, PPARs are now also being investigated as potential anti-NASH druggable targets. In this paper, we review the mechanisms of action and physiological functions of PPARs and discuss the position of the different PPAR agonists in the therapeutic landscape of NASH. We particularly focus on the PPAR agonists currently under evaluation in clinical phase II and III trials. Preclinical strategies and how refinement and optimization may improve PPAR-targeted anti-NASH drug testing are also discussed. Finally, potential caveats related to PPAR agonism in anti-NASH therapy are stipulated.

Transcriptomics data of a human in vitro model of non-alcoholic steatohepatitis exposed to elafibranor.

The present dataset contains the transcriptomic characterization of a novel in vitro model of non-alcoholic steatohepatitis (NASH) as well as its transcriptomics read-outs for the evaluation of elafibranor, a potential anti-NASH compound. We report whole genome microarray data (Affymetrix HG U133 plus 2.0) of human multipotent stem cell-derived hepatic cells (hSKP-HPC) exposed to mediators of NASH. These cells were exposed to lipogenic inducers (insulin, glucose, fatty acids) and pro-inflammatory factors (IL-1ß, TNF-α, TGF-ß) to trigger hepatocellular responses characteristic of NASH. In addition, to evaluate the anti-NASH features of elafibranor, a dual peroxisome proliferator-activated receptor (PPAR) agonist that currently is under investigation as a potential anti-NASH therapeutic, was tested this in vitro set-up. This paper provides a detailed description of the microarray data as well as an indication of their value for evaluating cell signaling pathways (e.g. NFκB network) during the in vitro evaluation of anti-NASH compounds. Raw microarray data of different testing conditions were deposited as.CEL files in the Gene Expression Omnibus of NCBI with GEO Series accession number GSE126484. Further interpretation and discussion of these data can be found in the corresponding research article (DOI: 10.1016/j.phrs.2019.04.016) Boeckmans et al., 2019.

Technological advancements for the development of stem cell-based models for hepatotoxicity testing.

Stem cells are characterized by their self-renewal capacity and their ability to differentiate into multiple cell types of the human body. Using directed differentiation strategies, stem cells can now be converted into hepatocyte-like cells (HLCs) and therefore, represent a unique cell source for toxicological applications in vitro. However, the acquired hepatic functionality of stem cell-derived HLCs is still significantly inferior to primary human hepatocytes. One of the main reasons for this is that most in vitro models use traditional two-dimensional (2D) setups where the flat substrata cannot properly mimic the physiology of the human liver. Therefore, 2D-setups are progressively being replaced by more advanced culture systems, which attempt to replicate the natural liver microenvironment, in which stem cells can better differentiate towards HLCs. This review highlights the most recent cell culture systems, including scaffold-free and scaffold-based three-dimensional (3D) technologies and microfluidics that can be employed for culture and hepatic differentiation of stem cells intended for hepatotoxicity testing. These methodologies have shown to improve in vitro liver cell functionality according to the in vivo liver physiology and allow to establish stem cell-based hepatic in vitro platforms for the accurate evaluation of xenobiotics.

Elafibranor restricts lipogenic and inflammatory responses in a human skin stem cell-derived model of NASH.

Non-alcoholic steatohepatitis (NASH) is characterized by hepatocellular steatosis with concomitant hepatic inflammation. Despite its pandemic proportions, no anti-NASH drugs have been approved yet. This is partially because drug development is decelerated due to the lack of adequate tools to assess the efficacy of potential new drug candidates. The present study describes the development and application of a new preclinical model for NASH using hepatic cells generated from human skin-derived precursors. Exposure of these cells to lipogenic (insulin, glucose, fatty acids) and pro-inflammatory factors (IL-1ß, TNF-α, TGF-ß) resulted in a characteristic NASH response, as indicated by intracellular lipid accumulation, modulation of NASH-specific gene expression, increased caspase-3/7 activity and the expression and/or secretion of inflammatory markers, including CCL2, CCL5, CCL7, CCL8, CXCL5, CXCL8, IL1a, IL6 and IL11. The human relevance of the proposed NASH model was verified by transcriptomics analyses that revealed commonly modulated genes and the identification of the same gene classes between the in vitro system and patients suffering from NASH. The application potential of this in vitro model was demonstrated by testing elafibranor, a promising anti-NASH compound currently under clinical phase III trial evaluation. Elafibranor attenuated in vitro key features of NASH, and dramatically lowered lipid load as well as the expression and secretion of inflammatory chemokines, which in vivo are responsible for the recruitment of immune cells. This reduction in inflammatory response was NFκB-mediated. In summary, this human-relevant, in vitro system proved to be a sensitive testing tool for the investigation of novel anti-NASH compounds.

Human-based systems: Mechanistic NASH modelling just around the corner?

Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by excessive triglyceride accumulation in the liver accompanied by inflammation, cell stress and apoptosis. It is the tipping point to the life-threatening stages of non-alcoholic fatty liver disease (NAFLD). Despite the high prevalence of NASH, up to five percent of the global population, there are currently no approved drugs to treat this disease. Animal models, mostly based on specific diets and genetic modifications, are often employed in anti-NASH drug development. However, due to interspecies differences and artificial pathogenic conditions, they do not represent the human situation accurately and are inadequate for testing the efficacy and safety of potential new drugs. Human-based in vitro models provide a more legitimate representation of the human NASH pathophysiology and can be used to investigate the dysregulation of cellular functions associated with the disease. Also in silico methodologies and pathway-based approaches using human datasets, may contribute to a more accurate representation of NASH, thereby facilitating the quest for new anti-NASH drugs. In this review, we describe the molecular components of NASH and how human-based tools can contribute to unraveling the pathogenesis of this disease and be used in anti-NASH drug development. We also propose a roadmap for the development and application of human-based approaches for future investigation of NASH.