"Introduction. Through guided differentiation, human-induced pluripotent stem cells (hiPSCs) offer a reliable and unlimited source of hepatocytes that are certainly proving themselves useful in numerous tissue engineering and personalized medicine applications. However, concerns persist regarding the lack of functional maturity and long-term maintenance in culture of hiPSC-derived hepatocytes (iHeps). Indeed, iHeps still display a fetal hepatic phenotype rather than an adult one and, in particular, they continue to express fetal markers such as α-fetoprotein (AFP), as well as being poor at reproducing key mature functions, such as the activity of many detoxification enzymes. Improved protocols and 3D culture systems have shown that the hepatic functions of iHeps can be enhanced, thus contributing to the study of liver development, diseases and the efficacy of medicinal products, but hepatocytes with adult features had not yet been generated. We are now very close to generate such hepatocytes, self-assembled as organoids (iHep-Orgs).
Methodology. During this study, we self-assembled hiPSC-derived liver progenitors and refined the final step of our differentiation protocol to generate hepatocyte organoids (iHep-Orgs). Alongside hepatocyte growth factor (HGF), the iHep-Orgs were exposed to vitamin K and were supplied daily with changing concentrations of dexamethasone (Dex); moreover, oncostatin M (OSM) concentrations were gradually tapered until complete removal.
Results. The refinements made to our protocol combined with 3D culture allowed the generation of iHep-Orgs proved to be very similar in function to PHH-Orgs. Worthy of attention was the complete disappearance of AFP expression and secretion by iHep-Orgs to the benefit of ALB secretion. Indeed, this is a hallmark widely used to discriminate fetal and adult hepatocytes, so the loss of AFP expression testifies them achieving a high functional maturity. Another important marker of the improved maturation of iHeps is the extinguished expression of the fetal enzyme CYP3A7 mRNA with the concurrent appearance of CYP3A4 mRNA expression, alongside the expression of the typical markers of hepatic differentiation. Finally, the ability of iHep-Orgs to fulfill some of the most important adult liver functions, further testifies to the robust and sustained maturation of our iHeps cultured as organoids. Bile acid production, in concentrations similar to those of PHHs, and their secretion proved that the hepatocytes within iHep-Orgs have also acquired the expected complex polarity. The distribution of the apical markers BSEP and MDR1, concomitant with the expression of ZO1, both on the surface of the organoids and into the structures suggested indeed the presence of a developed bile canalicular network.
Finally, iHep-Orgs were able to fulfill some of the most important adult liver functions, such as phase I and II metabolisms, alcohol metabolism, a hormone-induced response to hyperglycemic and hypoglycemic conditions, as well as a remarkable detoxification ability. Conclusions. Overall, the iHep-Orgs we generated represent a homogenous cell population with close similarity to primary adult hepatocytes and might represent an important tool for the development of new liver models for either liver diseases or pharmaco-toxicology studies, tissue engineering, and regenerative applications.
Financial support: RHU program “iLite” (Innovations for Liver Tissue Engineering) granted by PIA2 through ANR-16-RHUS-0005"