Reprogramming adult human hepatocytes into liver progenitors with unlimited self-renewal, efficient differentiation, and transplantation capacities

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The liver is the largest internal organ in the body and performs ~500 tasks, including detoxification of certain substances and balancing energy metabolism. While it has an excellent regeneration capacity, chronic damage due to years of alcohol abuse or high fat diets can result in severe liver function failure. Liver transplantation is currently the only available treatment for terminal liver diseases, but the liver transplant waiting list is getting longer in recent days due to the limited number of donors and the increasing number of patients. Hepatocytes, which make up 55-68% of the liver mass, are the major players of metabolism and drug biotransformation in our body, and the use of human hepatocyte for the toxicology tests in the laboratory can reduce the numbers of animals commonly used. It will also allow more precise prediction of the drug toxicity, because different species have different metabolic activities. However, mature hepatocytes cannot be propagated in the laboratory and their supply for the toxicology tests also depends on the limited number of donors. Excitingly, recent research has enabled the conversion of the normally non-proliferative state of mature hepatocytes from rat liver to a proliferative state by using a specific culture condition in the laboratory. These 'reprogrammed' cells are in a similar state to 'hepatocyte progenitors', which transiently proliferate and then become fully functional mature hepatocytes in the developing or regenerating liver in the body. Importantly, the artificially created rat hepatocyte progenitors showed an unlimited proliferation capacity and a capacity to go back to non-proliferative, fully functional mature hepatocytes when placed in another culture condition. If the same cell state conversion, called cellular reprogramming, is successfully achieved with human hepatocytes, we can generate an unlimited number of fully functional hepatocytes from one donor. However, human hepatocyte reprogramming with the same or similar modified conditions so far have resulted in only a limited success. The hepatocyte progenitors derived from the adult human liver could proliferate only <1 month. Those from the infant liver could gain an unlimited proliferation capacity, reflecting the highly proliferative nature of neonatal hepatocytes. However, they lost the capacity to make fully functional hepatocytes, called a differentiation capacity, after 2 weeks in the dish. In order to achieve successful reprogramming of human hepatocytes into the right progenitor state, we will use techniques called genetic engineering. With this technique, we will supply multiple candidate genes, which are missing in the partially reprogrammed non-functional human progenitors when compared to the fully functional rat progenitors, to the human progenitors. This would result in fully reprogrammed human hepatocyte progenitors with an unlimited proliferation and efficient differentiation capacities in a genetic modification-dependent manner. In parallel, we will look for environmental cues that can help to maintain the reprogrammed state in the absence of the genetic manipulation. This will be performed by eliminating each one of all ~20,000 genes in the genome simultaneously in millions of cells using the state-of-the-art technology called CRISPR/Cas9 genome editing. This strategy will inform us which environmental cues are essential to maintain the reprogrammed human hepatocyte progenitors, allowing us to supplement these essential components (protein/chemicals) in the culture condition, instead of the genetic modifications. In summary, this project aims to develop a strategy to generate fully functional human liver progenitors that will be an unlimited source of fully functional mature hepatocytes, used for drug screening, toxicology tests, as well as cell therapies, in the absence of genetic modifications.