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

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.

In 2017, the Ochiya group reprogrammed rat mature hepatocytes by the use of 3 small molecules, and generated liver progenitors with unlimited self-renewal and efficient differentiation capacities, called chemically induced liver progenitors (CLiPs). While several groups, including ours, have applied the same or similar culture conditions to human hepatocytes, so far only limited success was obtained. Human CLiPs (hCLiPs) derived from the adult human hepatocytes did not proliferate over 1 month. hCLiPs from the fetal/neonatal hepatocytes had an unlimited proliferation capacity, but lost their differentiation capacity within 2 weeks. In this project, 1) we aim to generate hCLiPs with unlimited proliferation and efficient differentiation capacities using doxycycline (Dox)-inducible shRNA and transcription factors (TF) expression based on the existing data sets. In this candidate approach, we have already found that down-regulation of a hepatocellular carcinoma (HCC) suppressor was sufficient to overcome the proliferation block in adult hCLiPs, and a cocktail of 6 TFs up-regulated ALBUMIN (ALB) expression in late passage adult hCLiPs. High ALB expression correlates with a CLiP state with a good differentiation capacity in rat, mouse and human. In parallel, 2) we will look for pathways essential for the maintenance of CLiP proliferation/self-renewal and differentiation capacities using CRISPR/Cas9-mediated genome-wide knockout screens with early passage mouse CLiPs and genetically engineered human CLiPs generated in 1). Finally, 3) we will apply the compounds that can activate the essential pathways identified in 2) to establish a culture condition that supports self-renewal of the fully functional hCLiPs upon removal of Dox using the cell lines established in 1). This work will not only provide an unlimited source of fully functional hepatocytes for toxicology tests and drug screens, but also pave a way to generate non-genetically modified hCLiPs for cell therapies.

In this project, we aim to generate hepatocyte progenitors, called chemically reprogrammed liver progenitors (CLiPs), with unlimited proliferation and efficient differentiation capacities by reprogramming human mature hepatocytes using a combination of exogenous transcription factors and small molecules. The outcome of the project would provide us with unlimited supply of fully functional hepatocytes, suitable for drug screening and toxicology tests in vitro.

Hepatocytes are important cell types for in vitro drug toxicology tests because they are the major source of drug metabolism and biotransformation in our body. However, freshly isolated hepatocytes, which have limited availability, quickly lose their functionality and cannot proliferate in vitro. Therefore, alternative sources of functional human hepatocytes have been sought. Unfortunately, currently available human ES/iPS cell-derived hepatocytes or induced hepatocytes (iHep) generated by transcription factor-mediated cell conversions are not fully functional. Human CLiPs we will generate in this project could overcome the shortage of human hepatocytes in toxicology tests.
Disease modelling is another area of research our human CLiPs could contribute. Recently a model for Non-alcoholic fatty liver disease (NAFLD) has been developed using freshly isolated human hepatocytes. NAFLD is one of the most common forms of liver disease worldwide, and 1 in 3 people in the UK present with an early stage form of NAFLD. Thus, the in vitro NAFLD model with human hepatocytes is a powerful tool for better understanding of this disease and drug discovery. However, the need of human hepatocytes from the donated liver with limited availability was the bottleneck to use this in vitro model routinely. CLiP-derived fully functional hepatocyte from this study could replace the need of fresh hepatocytes.

Overall, the unlimited supply of functional human hepatocytes from human CLiPs could decrease the cost and time necessary to develop safe and effective drugs. Better understanding of hepatocyte biology overcoming the limited availability of the research materials would also contribute to medicine.

In addition, CLiP-derived hepatocytes can be a tool to develop hepatocyte transplantation (HT) therapy. Since 1970, deaths due to liver disease have increased by 400% in UK. Currently, over 40 people die from liver disease in the UK every day. This is in stark contrast to other major killer diseases, such as heart disease and cancer, in which the number of deaths have either remained stable or decreased in the past 50 years. HT has been already used to treat patients with genetic metabolic disorders, but not on acute and chronic liver diseases. Unlimited supply of fully functional human hepatocytes from human CLiPs can allows us to investigate which liver disease conditions are suitable for HT using animal models. While human CLiPs we will generate in this project will have genetic modifications, they will form a base to derive genetically unmodified human CLiPs, which could be suitable for HT in future.