Postdoctoral Researcher Federal University of Rio de Janeiro Rio de Janeiro , Rio de Janeiro, Brazil
Abstract: Acellular liver scaffolds (ALS) are powerful tools to overcome organ shortage problems. To date, it is unknown whether transplanted ALS are affected by cirrhotic livers, either becoming cirrhotic themselves or instead remaining as a robust template for healthy cell growth after transplantation (tx). Since Hepatic Stellate Cells (HSCs) play a key role in fibrogenic processes, the aim of this study was to analyse whether the change in the ECM impacts HSC behaviour (pro-fibrotic reverses into a more quiescent state?). To address this, in vivo and in vitro experiments using human 3D ALS were performed. For in vitro experiments, primary human HSC (250k) were cultured in cirrhotic ALS for 14 days. HSC were then extracted from cirrhotic ALS and reseeded in healthy ALS for 14 days. Histological analysis (H&E), cell viability, RT-PCR (Col1alpha1, PDGFR, ACTA2, and CYTGB), and western blot analysis were performed (n=4). For in vivo analysis, decellularized livers obtained from Wistar rats (Animal Care approval-UFRJ 097/20) were transplanted into cirrhotic recipient rats. Cirrhotic recipient rats (n=5) received 5% ethanol in drinking water and i.p injections of carbon tetrachloride (1 ml/Kg) for 8 weeks, underwent hepatectomy (10%) and partial ALS orthotopic tx. H&E staining, immunohistochemistry (alpha-SMA), and microcirculation analysis were performed. RT-PCR and western blot analysis from in vitro experiments revealed that the fibrotic HSC reverses to a more quiescent state when HSC are extracted from cirrhotic and subsequently seeded and cultured in healthy ALS. Results from in vivo analysis showed that HSC migrated from the recipient cirrhotic liver to healthy ALS after tx. Histological and microcirculation analyses revealed that HSC underwent remodeling, transitioning into a more quiescent state 30 days after tx. Our results showed that the ECM affected HSC behaviour, reversing from a profibrotic to a quiescent state both in vivo and in vitro. Currently, hepatocytes derived from induced pluripotent stem cells are cultured with HSC to investigate whether cell-cell and cell-ECM could affect HSC behaviour. Thus, 3D ALS represents a robust template for healthy cell growth stimulation that can impact on liver regeneration after hepatectomy.
Funding Source: CNPq, Capes, FAPERJ, INCT-REGENERA, Ministério da Saúde.
