Human skin stem cell-derived hepatic cells as in vitro drug discovery model for insulin-driven de novo lipogenesis.

Metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease (NAFLD), is characterized by intrahepatic triglyceride accumulation and can progress to metabolic dysfunction-associated steatohepatitis (MASH) and liver fibrosis. Hepatic de novo lipogenesis (DNL), activated by glucose and insulin, is a central pathway contributing to early-stage development of MASLD. The emerging global prevalence of MASLD highlights the urgent need for pharmaceutical intervention to combat this health threat. However, the identification of novel drugs that could inhibit hepatic DNL is hampered by a lack of reliable, insulin-sensitive, human, in vitro, hepatic models. Here, we report human skin stem cell-derived hepatic cells (hSKP-HPC) as a unique in vitro model to study insulin-driven DNL (iDNL), evidenced by both gene expression and lipid accumulation readouts. Insulin-sensitive hSKP-HPC showed increased sterol regulatory element-binding protein 1c (SREBP-1c) expression, a key transcription factor for DNL. Furthermore, this physiologically relevant in vitro human steatosis model allowed both inhibition and activation of the iDNL pathway using reference inhibitors and activators, respectively. Optimisation of the lipid accumulation assay to a high-throughput, 384-well format enabled the screening of a library of annotated compounds, delivering new insights on key players in the iDNL pathway and MASLD pathophysiology. Together, these results establish the value of the hSKP-HPC model in preclinical development of antisteatotic drugs to combat MASLD.

From NAFLD to MAFLD: Aligning Translational In Vitro Research to Clinical Insights.

Although most same-stage non-alcoholic fatty liver disease (NAFLD) patients exhibit similar histologic sequelae, the underlying mechanisms appear to be highly heterogeneous. Therefore, it was recently proposed to redefine NAFLD to metabolic dysfunction-associated fatty liver disease (MAFLD) in which other known causes of liver disease such as alcohol consumption or viral hepatitis do not need to be excluded. Revised nomenclature envisions speeding up and facilitating anti-MAFLD drug development by means of patient stratification whereby each subgroup would benefit from distinct pharmacological interventions. As human-based in vitro research fulfils an irrefutable step in drug development, action should be taken as well in this stadium of the translational path. Indeed, most established in vitro NAFLD models rely on short-term exposure to fatty acids and use lipid accumulation as a phenotypic benchmark. This general approach to a seemingly ambiguous disease such as NAFLD therefore no longer seems applicable. Human-based in vitro models that accurately reflect distinct disease subgroups of MAFLD should thus be adopted in early preclinical disease modeling and drug testing. In this review article, we outline considerations for setting up translational in vitro experiments in the MAFLD era and allude to potential strategies to implement MAFLD heterogeneity into an in vitro setting so as to better align early drug development with future clinical trial designs.

Transcriptional Profile of Cytokines, Regulatory Mediators and TLR in Mesenchymal Stromal Cells after Inflammatory Signaling and Cell-Passaging.

Adult human subcutaneous adipose tissue (AT) harbors a rich population of mesenchymal stromal cells (MSCs) that are of interest for tissue repair. For this purpose, it is of utmost importance to determine the response of AT-MSCs to proliferative and inflammatory signals within the damaged tissue. We have characterized the transcriptional profile of cytokines, regulatory mediators and Toll-like receptors (TLR) relevant to the response of MSCs. AT-MSCs constitutively present a distinct profile for each gene and differentially responded to inflammation and cell-passaging. Inflammation leads to an upregulation of IL-6, IL-8, IL-1ß, TNFα and CCL5 cytokine expression. Inflammation and cell-passaging increased the expression of HGF, IDO1, PTGS1, PTGS2 and TGFß. The expression of the TLR pattern was differentially modulated with TLR 1, 2, 3, 4, 9 and 10 being increased, whereas TLR 5 and 6 downregulated. Functional enrichment analysis demonstrated a complex interplay between cytokines, TLR and regulatory mediators central for tissue repair. This profiling highlights that following a combination of inflammatory and proliferative signals, the sensitivity and responsive capacity of AT-MSCs may be significantly modified. Understanding these transcriptional changes may help the development of novel therapeutic approaches.

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.

Inflammation Alters the Secretome and Immunomodulatory Properties of Human Skin-Derived Precursor Cells.

Human skin-derived precursors (SKP) represent a group of somatic stem/precursor cells that reside in dermal skin throughout life that harbor clinical potential. SKP have a high self-renewal capacity, the ability to differentiate into multiple cell types and low immunogenicity, rendering them key candidates for allogeneic cell-based, off-the-shelf therapy. However, potential clinical application of allogeneic SKP requires that these cells retain their therapeutic properties under all circumstances and, in particular, in the presence of an inflammation state. Therefore, in this study, we investigated the impact of pro-inflammatory stimulation on the secretome and immunosuppressive properties of SKP. We demonstrated that pro-inflammatory stimulation of SKP significantly changes their expression and the secretion profile of chemo/cytokines and growth factors. Most importantly, we observed that pro-inflammatory stimulated SKP were still able to suppress the graft-versus-host response when cotransplanted with human PBMC in severe-combined immune deficient (SCID) mice, albeit to a much lesser extent than unstimulated SKP. Altogether, this study demonstrates that an inflammatory microenvironment has a significant impact on the immunological properties of SKP. These alterations need to be taken into account when developing allogeneic SKP-based therapies.

The Impact of Cell-Expansion and Inflammation on The Immune-Biology of Human Adipose Tissue-Derived Mesenchymal Stromal Cells.

Background: As a cell-based therapeutic, AT-MSCs need to create an immuno-reparativeenvironment appropriate for tissue repair. In the presence of injury, MSCs may have to proliferate and face inflammation. Clinical application requires repeated administrations of a high number of cellswith a well-established immune profile. Methods: We have established an immuno-comparative screening by determining the expression of 28 molecules implicated in immune regulation. This screening was performed during cell-expansion and inflammatory priming of AT-MSCs. Results: Our study confirms that AT-MSCs are highly expandable and sensitive to inflammation. Both conditions have substantially modulated the expression of a panel of immunological marker. Specifically, CD34 expression was substantially decreased upon cell-passaging. HLA-ABC, CD40 CD54, CD106, CD274 and CD112 were significantly increased by inflammation. In vitro cell-expansion also significantly altered the expression profile of HLA-DR, CD40, CD62L, CD106, CD166, HLA-G, CD200, HO-1, CD155 and ULBP-3. Conclusion: This study points out the response and characteristics of MSCs following expansion and inflammatory priming. It will strength our knowledge about the molecular mechanisms that may improve or hamper the therapeutic potential of MSCs. These immunological changes need to be further characterized to guarantee a safe cellular product with consistent quality and high therapeutic efficacy.

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.

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.

Human Skin-Derived Precursor Cells: Isolation, Expansion, and Hepatic Differentiation.

Human skin-derived precursor cells are a multipotent stem cell population that resides within the dermis throughout adulthood. Human skin-derived precursor cells can be isolated, purified, and expanded in large quantities from any patient, in health and disease, and differentiated to mesodermal and ectodermal cell types. Recently, it was also found that they can be directed towards hepatic cells with acquired properties of toxicological relevance. As such, they represent a valuable cell source for the further development of human-relevant in vitro models for the identification and quantification of hepatotoxic compounds. In this chapter, a robust basic methodology to isolate, expand, and differentiate human skin-derived precursor cells into hepatic cells in a sequential and time-dependent way is provided.

Measurement of Albumin Secretion as Functionality Test in Primary Hepatocyte Cultures.

One of the most important functions of hepatocytes is the synthesis of serum proteins, more specifically of serum albumin. Albumin secretion in serum is essential, since it maintains the oncotic pressure in the body. Measurement of albumin secretion is used as a liver function test to indicate potential liver injury and liver pathology. In this chapter, a protocol for the measurement of albumin secretion in the supernatant of cultured rat hepatocytes is described. The procedure relies on an enzyme-linked immunosorbent assay allowing rat albumin to be quantitatively measured.

Evaluation of a new standardized enzymatic isolation protocol for human umbilical cord-derived stem cells.

The umbilical cord (UC) represents an important source of mesenchymal stem cells (MSC). These human UC-derived MSC (UC-MSC) have already been isolated using a protocol based on the migratory and plastic adhesive properties of MSC (UC-MSC-Mig). The UC-MSC-Mig isolation method, however, is difficult to standardize. Therefore, we developed an enzymatic isolation protocol (UC-MSC-Enz) to overcome the above mentioned disadvantages. First, we investigated the UC-MSC-Enz for their MSC properties. We found that UC-MSC-Enz express the MSC markers CD73, CD90 and CD105 and are able to differentiate into osteoblasts, adipocytes and chondroblasts fulfilling the MSC criteria of the International Society for Cellular Therapy. Previously we found that UC-MSC-Mig are unique among MSCs due to their significant expression of several hepatic (progenitor) markers. Therefore, we also investigated the expression of hepatic transcription factors and other hepatic markers in UC-MSC-Enz at both the mRNA and protein level. We found that the expression of hepatic transcription factors (GATA4, GATA6, SOX9 and SOX17) and hepatic markers (AFP, DPP4, CX43, DKK1, DSG2, KRT18 and KRT19) in UC-MSC-Enz was not significantly different from those of UC-MSC-Mig. Consequently, this optimized enzyme-based method represents a fast, robust and standardized way to isolate UC-MSC for a broad range of applications.

Proliferative and phenotypical characteristics of human adipose tissue-derived stem cells: comparison of Ficoll gradient centrifugation and red blood cell lysis buffer treatment purification methods.

BACKGROUND AIMS: Adult human subcutaneous adipose tissue harbors a multipotent stem cell population, the so-called human adipose tissue-derived mesenchymal stromal cells (AT-MSCs). These cells are able to differentiate in vitro into various cell types and possess immunomodulatory features. Yet procedures to obtain AT-MSCs can vary significantly. The two most extensively used AT-MSC purification techniques are (i) density gradient centrifugation using Ficoll and (ii) red blood cell (RBC) lysis buffer treatment of the stromal vascular fraction. In the context of potential clinical cell therapy, the stem cell yield after purification and upon consecutive passages, as well as the purity of the obtained cell population, are of utmost importance. METHODS: We investigated the expansion capacity and purity of AT-MSCs purified by both procedures immediately after isolation and upon consecutive passages. We also investigated possible purification-dependent differences in their expression of immune-inhibitory factors and cell adhesion molecules. RESULTS: We found that RBC lysis buffer treatment is a more robust and easier method to purify AT-MSCs than density gradient fractionation. However, the resulting AT-MSC-RBC population contains a significantly higher number of CD34(+) cells, particularly during the first passages after plating. From passage 4 onward, no significant differences could be observed between both populations with respect to the immunophenotype, expansion capacity and expression of immune inhibitory factors and cell adhesion molecules. CONCLUSIONS: Our data show that RBC lysis buffer treatment may be a good alternative to density fractionation, providing a faster, more robust and easier method to purify AT-MSCs with biologically preserved characteristics.

Characterization of hepatic markers in human Wharton's Jelly-derived mesenchymal stem cells.

Stem cell technology could offer a unique tool to develop human-based in vitro liver models that are applicable for testing of potential liver toxicity early during drug development. In this context, recent research has indicated that human Wharton's Jelly-derived mesenchymal stem cells (hWJs) represent an interesting stem cell population to develop human hepatocyte-like cells. Here, an in-depth analysis of the expression of liver-specific transcription factors and other key hepatic markers in hWJs is evaluated at both the mRNA and protein level. Our results reveal that transcription factors that are mandatory to acquire and maintain an adult hepatic phenotype (HNF4A and HNF1A), as well as adult hepatic markers (ALB, CX32, CYP1A1, CYP1A2, CYP2B6 and CYP3A4) are not expressed in hWJs with the exception of K18. On the contrary, transcription factors involved in liver development (GATA4, GATA6, SOX9 and SOX17) and liver progenitor markers (DKK1, DPP4, DSG2, CX43 and K19) were found to be highly expressed in hWJs. These findings provide additional indication that hWJs could be a promising stem cell source to generate hepatocyte-like cells necessary for the development of a functional human-based in vitro liver model.

Mesoderm-derived stem cells: the link between the transcriptome and their differentiation potential.

Human adult stem cells (hASCs) have become an attractive source for autologous cell transplantation, tissue engineering, developmental biology, and the generation of human-based alternative in vitro models. Among the 3 germ cell layers, the mesoderm is the origin of today's most widely used and characterized hASC populations. A variety of isolated nonhematopoietic mesoderm-derived stem cell populations exist, and all of them show important differences in terms of function, efficacy, and differentiation potential both in vivo and in vitro. To better understand whether the intrinsic properties of these cells contribute to the overall differentiation potential of hASCs, we compared the global gene expression profiles of 4 mesoderm-derived stem cell populations: human adipose tissue-derived stromal cells, human bone marrow-derived stromal cells (hBMSCs), human (fore)skin-derived precursor cells (hSKPs), and human Wharton's jelly-derived mesenchymal stem cells (hWJs). Significant differences in gene expression profiles were detected between distinct stem cell types. hSKPs predominantly expressed genes involved in neurogenesis, skin, and bone development, whereas hWJs and, to some extent, hBMSCs showed an increased expression of genes involved in cardiovascular and liver development. Interestingly, the observed differential gene expression of distinct hASCs could be linked to existing differentiation data in which hASCs were differentiated toward specific cell types. As such, our data suggest that the intrinsic gene expression of the undifferentiated stem cells has an important impact on their overall differentiation potential as well as their application in stem cell-based research. Yet, the factors that define these intrinsic properties remain to be determined.