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ENHANCE Report Summary

Project ID: 705607
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - ENHANCE (Engineering the 3D Niche to enHANCE functionality of hepatocytes derived from human iPSCs)

Reporting period: 2016-03-01 to 2018-02-28

Summary of the context and overall objectives of the project

Liver disease is a growing clinical burden, resulting in over 100,000 deaths and around 6,000 liver transplants each year in Europe. Where there is the possibility of natural regeneration of the liver, auxiliary liver transplantation with a small amount of donor tissue can support hepatic function during recovery. However, the availability of liver transplantation therapies is now severely limited by insufficient organ donation, and similarly the supply of primary human hepatocytes is very restricted and since these cells cannot be easily expanded in vitro while retaining their characteristics.
Hepatocytes derived from human induced pluripotent stem cells (i-Heps) afford us the exciting possibility of redressing these growing shortages, and heralding in a new era of patient specific drug and disease modelling.
Clinical applications are restricted by their poor in vivo functionality, in comparison with the gold standard-freshly isolated primary hepatocytes. This insufficiency, due largely to incomplete maturation, also limits their usefulness for other target applications such as in vitro disease modelling, drug, toxicology, and developmental studies.
One of the major reasons for this failure is the lack of a systematic approach, empirically defining a signature of maturity that must be reached in order for i-Heps to cross a threshold of usefulness.
Emerging evidence suggests the three dimensional environment surrounding a cell (the niche) critically influences cellular function.
The objective of ENHANCE was to identify relevant factors from the extracellular niche which promote i-Heps function in 3D and use this technology to generate functionally optimised i-Heps constructs suitable for clinical transplantation.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

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 th

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

To our knowledge, this is the first attempt to produce liver organoids using just a combination of iPSC-derived hepatic progenitors and a synthetic hydrogel. The unique modular features of the ICC scaffold will allow study of the complex, combinatorial influences of physical and chemical signals during liver organogenesis in a physiologically relevant, dissectible 3D microenvironment. In addition, the scalable structure and clinically compliant materials employed open up the possibility of use for human therapy. These results highlight the enormous potential of bio-engineered organoids for discovery and translational science.
This research area is directly relevant to public health problem because hiPSC derived liver products offer the possibility of redressing the growing shortage in hepatic transplantation tissue providing a new tool for the study of liver diseases and drug hepatotoxicity. Cell therapy products identified through this research are currently under IP generation.

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