Abstract
BackgroundLiver 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 iPS-derived cells could represent great challenges for a translational perspective.MethodsHere, we evaluated the influence of isogenic versus heterogenic non-parenchymal cells, using iPS-derived 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.ResultsWe 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.ConclusionAiming 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.
Highlights
Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies
The first attempts to bioengineer complex liver organoids (LOs) used hepatoblasts derived from human pluripotent stem cells in conjunction with primary human non-parenchymal cells (NPC), such as human umbilical cord-derived endothelial cells (HUVEC) and adipose tissue-derived mesenchymal stem cells (MSCs), all derived from different donors [1]
Results induced pluripotent stem cells (iPS) cell differentiation Aiming a broad applicability of our studies and reproducibility of the results, we carried out the experiments with three independent iPS cell lines
Summary
Liver organoid technology holds great promises to be used in large-scale population-based drug screening and in future regenerative medicine strategies. 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. The first attempts to bioengineer complex liver organoids (LOs) used hepatoblasts derived from human pluripotent stem cells (iPS) in conjunction with primary human NPC, such as human umbilical cord-derived endothelial cells (HUVEC) and adipose tissue-derived mesenchymal stem cells (MSCs), all derived from different donors [1]. Some other groups reported a series of combined protocols for generating isogenic LOs obtained from whole iPS-derived cells, obtained from the same donor, or by using primary NPCs from the same donor [7,8,9]. Takebe and collaborators [7] successfully generated LOs from human donors that could potentially be applied for high-throughput personalized screening of liver toxicity
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