Evidence of Adult Features and Functions of Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells and Self-Organized as Organoids

Abstract

Background: Human-induced pluripotent stem cell-derived hepatocytes (iHeps) have been shown to have considerable potential in liver diseases, toxicity, and pharmacological studies. However, there is a growing need to obtain iHeps that are truly similar to primary adult hepatocytes in terms of morphological features and functions. We generated such human iHeps, self-assembled as organoids (iHep-Orgs). Methods: iPSC-derived hepatoblasts were self-assembled into spheroids and differentiated into mature hepatocytes modulating final step of differentiation. Results: In about four weeks of culture, the albumin secretion levels and the complete disappearance of α-fetoprotein from iHep-Orgs suggested the acquisition of a greater degree of maturation than those previously reported. The expression of apical transporters and bile acid secretion evidenced the acquisition of complex hepatocyte polarity as well as the development of a functional and well-defined bile canalicular network confirmed by computational analysis. Activities recorded for CYP450, UGT1A1, and alcohol dehydrogenase, response to hormonal stimulation, and glucose metabolism were also remarkable. Finally, iHep-Orgs displayed a considerable ability to detoxify pathological concentrations of lactate and ammonia. Conclusions: With features similar to those of primary adult hepatocytes, the iHep-Orgs thus produced could be considered as a valuable tool for the development and optimization of preclinical and clinical applications.

Functional Evaluation of 3D Liver Models Labeled with Polysaccharide Functionalized Magnetic Nanoparticles

“…Therefore, 3D culture techniques have been widely used in drug discovery, medicine, and cosmetics. The human body is composed of various cells, among which the cells of the liver play an essential role in metabolism, detoxification, bile production, and secretion [ 4 , 5 , 6 , 7 , 8 , 11 , 12 , 13 , 14 , 15 , 39 , 40 , 41 ]. In order to investigate the effect of nanomaterials on the liver, a 3D liver model with liver-specific functions must be created in vitro .…”

Microencapsulated Hepatocytes Differentiated from Human Induced Pluripotent Stem Cells: Optimizing 3D Culture for Tissue Engineering Applications

“…The aggregation of iHBs was achieved via a self-assembly process after their seeding in agarose microwells, as previously reported [ 15 ]. An equivalent of 2.5 × 10 5 iHBs harvested at Day 11 of differentiation were re-suspended in 200 µL HCM and added to each well and then incubated at 37 °C under 5% CO 2 for 1 h before adding the culture medium supplemented with growth factors.…”

“…At Day 11 of differentiation, the iHBs thus obtained were detached using StemPro Accutase cell dissociation solution (Gibco). Whatever culture system was used, the iHBs were treated according to the differentiation protocol described by Messina et al [ 15 ], using HCM TM Hepatocyte Complete Medium (Lonza, Walkersville, MD, USA) supplemented with 20 ng/mL HGF, 0.1 ng/mL Dexamethasone (Dex) and 20 ng/mL Oncostatin M (OSM), and refreshed every second day until Day 22. On Day 18, in addition to the factors mentioned above, 10 ng/mL vitamin K (VK) (Roche, Basel, Switzerland) was added to the culture medium until the end of differentiation.…”

“…One strategy that may improve hepatocyte differentiation and functionality is 3D culture, as it both maximizes cell–cell interactions and approximates the physiological environment of the cells [ 14 ]. Indeed, 3D culture has been shown to play a major role in hepatocyte maturation when used for the differentiation of hiPSCs [ 15 , 16 , 17 ]. Cell encapsulation technology in alginate beads is one of the 3D culture methodologies that might represent an interesting tissue engineering approach appropriate for hepatocyte maturation.…”

In vitro long term differentiation and functionality of three-dimensional bioprinted primary human hepatocytes: application for in vivo engraftment

“…However, the use of hepatic cell lines is limited by their dedifferentiation compared from the human hepatocyte phenotype, and cells of animal origin are unrepresentative due to interspecies differences. iPSCs represents an important hope in the generation of human hepatocytes, it should be noted that their differentiation has long remained limited to the fetal stage, and their full differentiation to a hepatocyte level, requiring a long and complex protocol, has been only recently demonstrated [38]. Despite the promising advantages of bioprinting to produce advanced structures for the 3D culture of hepatocytes, to date there is no easily available model of PHH bioprinting.…”