Through imaging-based characterization (HCA) of freshly isolated hepatocytes from seventeen human donors, we devised and validated a novel algorithm for comparing the hepatic properties of different cells (Hepatocyte Likeness Index, HLI) relative to physiological standards. The method was then applied in a HCA screen of liver ECM proteins and niche factors to identify substrates that drive i-Heps closer to primary hepatocytes. The top hit, Laminin 411, was validated in two additional iPS lines, primary tissue and in an in-vitro model of alpha-1 antitrypsin deficiency. Cumulatively, these data underscore the importance of combining substrates, soluble factors and HCA pipelines in furthering iPS applications.
Results from the screen performed highlighted the important role played by Laminins, a group of heterotrimeric ECM proteins, in the hepatic progenitor response. This association had previously been reported in adult liver (Kallis et al., 2011) and in cholangiocyte differentiation (Takayama et al., 2016) but not in embryonic hepatocyte development. Here we demonstrated Laminin 411 to be a biologically relevant and important factor in advancing iPS hepatocyte differentiation and in human hepatic foetal development. This heterogeneity opens up the likelihood of downstream function being a consequence of co-engagement with a combination of as yet poorly defined soluble and insoluble factors. In the meantime, as shown by our A1AT drug-screening data, information from even the most basic of screens using the new algorithm can rapidly be translated into meaningful advances which in turn suggests our approach could be of widespread utility in the stem cell biology field. Lrrc17 and CYR61 are two f ECM proteins that when combined with Laminin 411 are improving i-HEPS albumin expression applying our HLI and then they are validated by ELISA and qPCR. We investigated their importance in human and mouse liver tissue during development using RNA-ISH technique. Results confirmed that those two proteins are highly expressed in foetal livers from human and mice, so they play an essential role in liver maturation.
iPSC derived organoids offer exciting possibilities in developmental biology, disease modelling and cell therapy. Realisation of those promises is hampered by requirements for Matrigel, which is poorly characterized, highly variable and of mouse origin (Fatehullah et al., 2016). A bio-engineered substitute is therefore essential and was recently reported for intestinal organoid generation (Gjorevski et al., 2016). A similar bottom up engineering approach for liver organoid production is urgently needed. Along those lines we recently developed a novel hepatocyte culture system composed of a 3D-hexagonally arrayed inverted colloidal crystal (ICC) scaffold (Shirahama et al., 2016). iPSC-derived hepatocytes (Rashid et al., 2010), known to be a good approximation to fetal hepatocytes, were used to produce scalable, interconnected hepatic organoids. Having confirmed the organoid’s maturation and disease modelling we next sought to explore the effects of in vivo transplantation. A pocket on the caudate lobe of murine liver was created by making an incision in the liver capsule. Organoids were placed into this pocket and sandwiched in place between the left lobe and the lower caudate lobe in order to achieve a bona fide homeostatic environment (Figure 1I). After 30 days, grafts were retrieved for further analysis. H&E staining revealed implants were well integrated into the host parenchyma, without evidence of significant fibrosis / inflammation whilst neo vascularization had successfully occurred between host and donor tissues. Histochemical staining with human albumin confirmed the implanted structures were of human origin, the organoid structure had remained intact and the presence of human albumin in host serum suggested cells remained functional.
Organoid generation by this approach is found to occur in a two-step process that recapitulates mammalian organogenesis with the resultant organoid structures demonstrating cellular zonation as previously reported in both embryonic and regenerating human liver buds. With respect to drug metabolism, protein secretion and disease modelling, IH -ICC organoids are both transcriptionally and functionally closer to adult liver tissue than controls. Physiological relevance is confirmed by capacity to integrate, vascularize and function following transplantation into livers of immunodeficient mice.
