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The only cure for many inborn metabolic liver diseases is liver transplantation, limited by shortage of donors. Transplantation of genetically corrected hepatocytes is an attractive alternative, but is hampered by the low amplification potential of these cells in vitro. The derivation of liver cells from induced pluripotent stem cells (iPSC) holds promises to circumvent this difficulty. However, the generation of iPSC induces genetic and epigenetic anomalies. This both decreases the efficiency of reprogramming and re-differentiation of these cells and could potentially result in long-term complications, including oncogenic transformation after re-implantation.

Therefore our project had 5 main objectives to assess the efficiency of an iPSC-based therapy for liver metabolic diseases:

1) Reprogramming of patient-specific CNI-HEP into iPSC.
2) Correction of CNI-iPSC by ex vivo lentiviral transduction.
3) Large-scale amplification of the corrected CNI-iPSC in vitro to obtain relevant cell numbers and differentiation of the corrected CNI-iPSC to HEP-like cells.
4) Epigenetic analysis of patient CNI-HEP, CNI-iPSC and differentiated-corrected HEP-like cells (HLC).
5) Safety and efficiency preclinical assessment of the corrected CNI-HEP-like cells by infusion into the liver of the CNI animal model.

During this period we have obtained relevant results for the first three objectives (mid-term report) plus we have recently developed a novel method for inducing proliferative hepatic progenitor cells (iHPC) from human hepatocytes as an alternative expandable cell source for liver therapy (Fig 1, in attachment).

Our results show that patient-specific iPSC can be generated from human hepatocytes as a proliferative cell source for the treatment of inborn monogenic diseases. However, primary hepatocytes might not be the ideal origin for reprogramming due to the high rate of defective clones obtained compared to other cell sources such as fibroblasts or blood cells (Unzu et al. Stem Cells International. Jan 2016).

Thus, we have developed a pharmacological protocol to generate proliferative hepatic progenitors (iHPC) from human hepatocytes. De-differentiation of primary hepatocytes to iHPC was achieved in less than 7 days by culturing the cells in medium with a cocktail of growth factors and small molecules. RNA sequencing of iHPC demonstrated that they displayed far more subtle changes in both transcriptome and TE expression, compared to hepatocyte-derived iPSC. The transcriptome study also revealed interesting similarities between both hepatocyte reprogramming to pluripotency and dedifferentiation. In addition, a DNA-array for detecting mutations during both processes was also performed showing higher number of mutations during iPSC generation than iHPC (Figures 2 and 3, in attachment).

Finally, in order to address the safety of iHPC transplantation, cells were injected into the liver of immuno-deficient mice showing iHPC differentiation in vivo and without triggering detectable tumor development. (Figure 4, in attachment).

Given the potential of this promising novel protocol and its technical and biological simplicity compared to reprogramming to pluripotency, we have filed an european patent for protecting the intellectual property on the method (EP 16174787.8) and a paper capitalizing on these results has been submitted to Nature Cell Biology.

Compared to iPSC generation from the same cell source, our results show that this method is safer, faster, cheaper and has a enormous socio-economic potential: Not only it could be used for liver cell therapy treatment, but also it could provide an important cell source for personalized drug testing. Pharmaceutical companies predict drug metabolic stability, liver toxicity or drug clearance in patients based on rodent animal models or in vitro assays using enzyme extractions from human cells. With this method we envision to accelerate the use of human hepatocytes for personalized drug metabolism screening. Only a minimal amount of patient’s cells will be required, which will tremendously facilitate the creation of bio-banks with primary cells of different ages, ethnicity or gender. Cells will be expanded for characterization and drug effect screening in human settings, allowing for a personalized analysis of drug metabolic stability, liver toxicity and drug clearance according to the different sub-groups.

In summary, our data confirm the remarkable plasticity of human hepatocytes and provide a promising methodological lead for the treatment of inborn hepatic diseases, for the development of a bio-artificial liver and for the personalized medicine of the future.

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