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The liver is responsible for many metabolic, endocrine and exocrine functions. Approximately 2 million deaths per year are associated with liver failure. Modern 3D bioprinting technologies allied with autologous induced pluripotent stem cells (iPS)-derived grafts could represent a relevant tissue engineering approach to treat end stage liver disease patients. However, protocols that accurately recapitulates liver’s epithelial parenchyma through bioprinting are still underdeveloped. Here we evaluated the impacts of using single cell dispersion (i.e. obtained from conventional bidimensional differentiation) of iPS-derived parenchymal (i.e. hepatocyte-like cells) versus using iPS-derived hepatocyte-like cells spheroids (i.e. three-dimensional cell culture), both in combination with non-parenchymal cells (e.g. mesenchymal and endothelial cells), into final liver tissue functionality. Single cell constructs showed reduced cell survival and hepatic function and unbalanced protein/amino acid metabolism when compared to spheroid printed constructs after 18 days in culture. In addition, single cell printed constructs revealed epithelial-mesenchymal transition, resulting in rapid loss of hepatocyte phenotype. These results indicates the advantage of using spheroid-based bioprinting, contributing to improve current liver bioprinting technology towards future regenerative medicine applications and liver physiology and disease modeling.

Mansonic schistosomiasis is an endemic disease in Brazil, with an estimated 10-12 million people infested. Among its clinical manifestations, the hepatosplenic form causes portal hypertension which, in turn, brings about severe digestive hemorrhage, the most serious complication of the disease. Normally, the patients are young, and have hepatosplenomegaly, hypersplenism without clinical manifestations, and slightly reduced hepatic function. The angiographic findings are characteristic, differing from those of hepatic cirrhosis. In Brazil, the definitive treatment for gastrointestinal hemorrhage is surgery, which should be done under elective conditions whenever possible. During a short period of time, known as the "risk period" (the time between the hemorrhagic episode and the surgery), propranolol has been used to prevent further bleeding. Surgical treatment is indicated only after the first episode, and never on a prophylactic basis. In 1977, a prospective, randomized trial was begun in order to assess the delayed results of the 3 surgical operations most widely used in this country. The study was interrupted after 94 patients had been operated on due to the high incidence of encephalopathy in the group who underwent classical splenorenal shunt. After a follow-up of at least 60 months and, at most, 130 months, the results showed that classical splenorenal shunt caused encephalopathy in 39.3% of the cases and distal splenorenal shunt in 14.8%. None of those submitted to esophagogastric devascularization with splenectomy developed encephalopathy. The 3 procedures showed similar rates of hemorrhagic recurrence.

Background: Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. Recently, some studies reported robust protocols for generating isogenic liver organoids using liver parenchymal and non-parenchymal cells derived from induced pluripotent stem cells (iPS) or using isogenic adult primary non-parenchymal cells. However, the use of whole iPSderived cells could represent great challenges for a translational perspective. Methods: Here, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPSderived or adult primary cell lines, in the liver organoid development. We tested four groups comprised of all different combinations of non-parenchymal cells for the liver functionality in vitro. Gene expression and protein secretion of important hepatic function markers were evaluated. Additionally, liver development-associated signaling pathways were tested. Finally, organoid label-free proteomic analysis and non-parenchymal cell secretome were performed in all groups at day 12. Results: We show that liver organoids generated using primary mesenchymal stromal cells and iPS-derived endothelial cells expressed and produced significantly more albumin and showed increased expression of CYP1A1, CYP1A2, and TDO2 while presented reduced TGF-β and Wnt signaling activity. Proteomics analysis revealed that major shifts in protein expression induced by this specific combination of non-parenchymal cells are related to integrin profile and TGF-β/Wnt signaling activity. Conclusion: Aiming the translation of this technology bench-to-bedside, this work highlights the role of important developmental pathways that are modulated by non-parenchymal cells enhancing the liver organoid maturation.

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