Human Liver Spheroids as a Model to Study Aetiology and Treatment of Hepatic Fibrosis

Non-alcoholic fatty liver disease affects approximately one billion adults worldwide. Non-alcoholic steatohepatitis (NASH) is a progressive disease and underlies the advancement to liver fibrosis, cirrhosis, and hepatocellular carcinoma, for which there are no FDA-approved drug therapies. We developed a hetero-cellular spheroid system comprised of primary human hepatocytes (PHH) co-cultured with crude fractions of primary human liver non-parenchymal cells (NPC) from several matched or non-matched donors, to identify phenotypes with utility in investigating NASH pathogenesis and drug screening. Co-culture spheroids displayed stable expression of hepatocyte markers (albumin, CYP3A4) with the integration of stellate (vimentin, PDGFRβ), endothelial (vWF, PECAM1), and CD68-positive cells. Several co-culture spheroids developed a fibrotic phenotype either spontaneously, primarily observed in PNPLA3 mutant donors, or after challenge with free fatty acids (FFA), as determined by COL1A1 and αSMA expression. This phenotype, as well as TGFβ1 expression, was attenuated with an ALK5 inhibitor. Furthermore, CYP2E1, which has a strong pro-oxidant effect, was induced by NPCs and FFA. This system was used to evaluate the effects of anti-NASH drug candidates, which inhibited fibrillary deposition following 7 days of exposure. In conclusion, we suggest that this system is suitable for the evaluation of NASH pathogenesis and screening of anti-NASH drug candidates.

Modulatory effects of TGF-β1 and BMP6 on thecal angiogenesis and steroidogenesis in the bovine ovary

Angiogenesis plays an integral role in follicular and luteal development and is positively regulated by several intra-ovarian factors including vascular endothelial growth factor A (VEGFA) and fibroblast growth factor 2 (FGF2). Various transforming growth factor-β (TGF-β) superfamily members function as intra-ovarian regulators of follicle and luteal function, but their potential roles in modulating ovarian angiogenesis have received little attention. In this study, we used a bovine theca interna culture model (exhibiting characteristics of luteinization) to examine the effects of TGF-β1 and bone morphogenetic protein 6 (BMP6) on angiogenesis and steroidogenesis. VEGFA/FGF2 treatment promoted endothelial cell network formation but had little or no effect on progesterone and androstenedione secretion or expression of key steroidogenesis-related genes. TGF-β1 suppressed basal and VEGFA/FGF2-induced endothelial cell network formation and progesterone secretion, effects that were reversed by an activin receptor-like kinase 5 (ALK5) inhibitor (SB-431542). The ALK5 inhibitor alone raised androstenedione secretion and expression of several transcripts including CYP17A1. BMP6 also suppressed endothelial cell network formation under VEGFA/FGF2-stimulated conditions and inhibited progesterone secretion and expression of several steroidogenesis-related genes under basal and VEGFA/FGF2-stimulated conditions. These effects were reversed by an ALK1/2 inhibitor (K02288). Moreover, the ALK1/2 inhibitor alone augmented endothelial network formation, progesterone secretion, androstenedione secretion and expression of several steroidogenesis-related genes. The results indicate dual suppressive actions of both TGF-β1 and BMP6 on follicular angiogenesis and steroidogenesis. Further experiments are needed to unravel the complex interactions between TGF-β superfamily signalling and other regulatory factors controlling ovarian angiogenesis and steroidogenesis.

Clinical ALK5 Inhibitor, Vactosertib, Reverses TGFβ-1 Stimulated Smad-2 Driven Ineffective Hematopoiesis in MDS

Transforming growth factor beta is an important regulator of hematopoiesis and its overactivation has been shown to occur in myelodysplastic syndromes. Even though TGF-beta leads to activation of the transcription factor SMAD2, the exact gene expression program and chromatin changes stimulated in human hematopoiesis are not well studied. We generated primary erythroid progenitors from human CD34+ stem cells and assessed genome occupancy of SMAD2 upon TGF-beta stimulation by Chip-seq analysis. We observed widespread occupancy of important stem cell and differentiation regulators with approximately 7000 binding regions. Parallel RNA-seq identified gene expression changes that are associated with smad2 binding at respective promoters and enhancers. Genes involved in stem cell maintenance as well as erythroid differentiation were prominently altered by TGF-beta and contained prominent SMAD2 peaks in hematopoietic progenitors. Next, to determine the relative contribution of various TGF-beta ligands in bone marrow failure, we analyzed a large cohort of MDS serum samples and found TGF-beta 1 (TGF-b1) to be the predominantly elevated ligand in 30% samples. Exposure to MDS patient serum led to erythroid differentiation blocks in primary hematopoietic stem cells in clonogenic cultures. Finally, we analyzed the effect of constitutive secretion of TGF-b1 in a transgenic mouse containing TGF-b1 gene driven by an albumin promoter. These mice constitutively secrete TGF-b1 and become cytopenic and show marrow histological changes that mimic human MDS. We observed stem cell expansion and block at the stage of common myeloid progenitor (CMP) in these mice; findings that are similar to stem and progenitors alterations seen in human MDS patients. To inhibit TGF-b1 signaling we next used a novel ALK5 (TGF-beta receptor I kinase) inhibitor that has been shown to be safe for human use. Vactosertib is specific small molecule inhibitor of ALK5 with a IC50 between 1 x 10-8 M and 1 x 10-9 M and has been shown to have an acceptable safety profile in phase I trial in solid tumors (NCT02160106). Vactosertib was able to inhibit smad2 phosphorylation and activation in a variety of hematopoietic cell lines. Vactosertib treatment led to reversal of hematopoietic alterations upon exposure to MDS serum in clonogenic assays. Vactosertib dosing in vivo also led to increased blood counts in TGF/alb transgenic mice and resulted in normalization of stem and progenitor changes. In conclusion, we demonstrate increased TGF-b1 levels in subset of MDS samples. We also determined TGF-b1 stimulated smad2 binding in important regulatory regions of the genome in primary human erythroid progenitors. We also demonstrate the preclinical efficacy of novel TGF-beta receptor 1 kinase inhibitor in vitro and in vivo. These studies provide preclinical rationale for using this ALK5 inhibitor in trials. Disclosures Huh: MedPacto Inc: Employment. Hwang:MedPacto Inc: Employment. Seong-Jin:MedPacto Inc: Employment. Will:Novartis Pharmaceuticals: Research Funding. Steidl:BayerHealthcare: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; GlaxoSmithKline: Research Funding; Celgene: Consultancy; Aileron Therapeutics: Consultancy, Research Funding; Stelexis Therapeutics: Equity Ownership, Membership on an entity's Board of Directors or advisory committees, Other: Scientific Co-Founder; Pieries Pharmaceuticals: Consultancy. Verma:Stelexis: Equity Ownership, Honoraria; Acceleron: Honoraria; Celgene: Honoraria; BMS: Research Funding; Janssen: Research Funding.

Molecular characterization of a precision-cut rat liver slice model for the evaluation of antifibrotic compounds

Precision-cut liver tissue slice (PCLS) contains all major cell types of the liver parenchyma and preserves the original cell-cell and cell-matrix contacts. It represents a promising ex vivo model to study liver fibrosis and test the antifibrotic effect of experimental compounds in a physiological environment. In this study using RNA sequencing, we demonstrated that various pathways functionally related to fibrotic mechanisms were dysregulated in PCLSs derived from rats subjected to bile duct ligation. The activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib each reversed a subset of genes dysregulated in fibrotic PCLSs, and of those genes we identified 608 genes whose expression was reversed by all three compounds. These genes define a molecular signature characterizing many aspects of liver fibrosis pathology and its attenuation in the model. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were further validated as efficacy end points for the evaluation of antifibrotic activity of experimental compounds. Finally, we showed that blockade of αV-integrins with a small molecule inhibitor attenuated the fibrotic phenotype in the model. Overall, our results suggest that the rat fibrotic PCLS model may represent a valuable system for target validation and determining the efficacy of experimental compounds. NEW & NOTEWORTHY We investigated the antifibrotic activity of three compounds, the activin receptor-like kinase-5 (Alk5) inhibitor SB525334, nintedanib, and sorafenib, in a rat fibrotic precision-cut liver tissue slice model using RNA sequencing analysis. A panel of 12 genes and 4 secreted biomarkers including procollagen I, hyaluronic acid (HA), insulin-like growth factor binding protein 5 (IGFBP5), and WNT1-inducible signaling pathway protein 1 (WISP1) were then established as efficacy end points to validate the antifibrotic activity of the αV-integrin inhibitor CWHM12. This study demonstrated the value of the rat fibrotic PCLS model for the evaluation of antifibrotic drugs.

A novel bioreactor technology for modelling fibrosis in human and rodent precision-cut liver slices

Summary boxWhat is already known about this subject?Currently there are no effective anti-fibrotic drugs to treat liver fibrosis and there is an urgent unmet need to increase our knowledge of the disease process and develop better tools for anti-fibrotic drug discovery.Preclinical in vitro cell cultures and animal models are widely used to study liver fibrosis and test anti-fibrotic drugs, but have shortfalls; cell culture models lack the relevant complex cell-cell interactions of the liver and animal models only reproduce some features of human disease.Precision Cut Liver Slices (PCLS) are structurally representative of the liver and can be used to model liver fibrosis and test anti-fibrotic drugs. However, PCLS are typically cultured in elevated, non-physiological oxygen levels and only have a healthy lifespan of 48h.What are the new findings?We have developed a novel bioreactor culture system that increases the longevity of functional PCLS to up to 6 days under normoxic conditions.Bioreactor cultured PCLS can be used to model fibrogenesis in both normal and fibrotic PCLS using a combination of biochemical and histological outputs.Administration of an Alk5 inhibitor effectively limits fibrogenesis in normal rodent and human PCLS and in rodent PCLS with established fibrosis.How might it impact on clinical practice in the foreseeable future?The extended longevity of bioreactor cultured PCLS represent a novel pre-clinical tool to investigate the cellular and molecular mechanisms of liver fibrosis.Bioreactor cultured human PCLS offer a clinically relevant system to test efficacy of anti-fibrotic drugs.AbstractObjectivePrecision cut liver slices (PCLS) retain the structure and cellular composition of the native liver and represent an improved system to study liver fibrosis compared to two-dimensional mono or co-cultures. The objective of this study was to develop a bioreactor system to increase the healthy lifespan of PCLS and model fibrogenesis.DesignPCLS were generated from normal rat or human liver, or 4-week carbon tetrachloride-fibrotic rat liver and cultured in our patented bioreactor. PCLS function was quantified by albumin ELISA. Fibrosis was induced in PCLS by TGFβ1 and PDGFββ stimulation. Alk5 inhibitor therapy was used. Fibrosis was assessed by fibrogenic gene expression, Picrosirius Red and αSmooth Muscle Actin staining, hydroxyproline assay and collagen 1a1, fibronectin and hyaluronic acid ELISA.ResultsBioreactor cultured PCLS are viable, maintaining tissue structure and stable albumin secretion for up to 6 days under normoxic culture conditions. Conversely, standard static transwell cultured PCLS rapidly deteriorate and albumin secretion is significantly impaired by 48 hours. TGFβ1 and PDGFββ stimulation of rat or human PCLS induced fibrogenic gene expression, release of extracellular matrix proteins, activation of hepatic myofibroblasts and histological fibrosis. Fibrogenesis slowly progresses over 6-days in cultured fibrotic rat PCLS without exogenous challenge. Alk5 inhibitor limited fibrogenesis in both TGFβ1 and PDGFββ stimulated PCLS and fibrotic PCLS.ConclusionWe describe a new bioreactor technology which maintains functional PCLS cultures for 6 days. Bioreactor cultured PCLS can be successfully used to model fibrogenesis and demonstrate efficacy of an anti-fibrotic therapy.