Decoding differentiation of cell lineages in the early human liver for application in stem cell differentiation and cell programming

The different types of human liver cell, such as hepatocytes and bile duct cells, are urgently needed in large quantities for regenerative medicine and for safety testing drugs in development. Adult cells are hard to come by and often vary in quality. Liver cancer cell lines are abnormal. Therefore, great interest has focused on turning stem cells in a dish into liver cells, or by reprogramming readily available cells (e.g. skin cells) directly into particular types of liver cell. However, despite nearly twenty years of trying our ability to do this is still limited. We think that knowing how these cells arise in early human development is the critical missing component. Recent advances in technology have revolutionised our ability to undertake these studies of how human organs are put together. We will build a framework of all the steps in how the early human liver developments by looking at some simple markers in tissue samples. We will then discover the full detail about the individual types of cell as they emerge by capturing every gene that they turn on. We have already shown that this type of work, when analysed using complex computer programmes, can pick out the most critical factors operating in each cell. We then have a distinctive opportunity to test these factors in stem cells and in cell reprogramming to see if they improve our ability to make liver cells that look more like the real thing. Once we have undertaken our work we will share both the results and the raw data with researchers around the world so that we can all benefit quickly from our new knowledge.

Human liver cell lineages are urgently needed in regenerative medicine and for drug safety assessment. Great interest has focused on differentiating human pluripotent stem cells (PSCs) or directly reprogramming fibroblasts in vitro. However, PSC-derived cells deviate from normal by the equivalent of early liver development. Reprogramming cell fate is still in its infancy, with a strong sense that we do yet have all the optimised factors. We hypothesise that in depth knowledge of early human liver development will transform these challenges.
Our objective is to provide comprehensive molecular understanding of the different hepatic lineages and how they arise. We will do this by defining a framework of early human liver development with key markers; highly practical new information for benchmarking PSC-derived cells in vitro. The markers will inform our next goal: to discover the full molecular phenotype of the critical liver cell-types as they arise. This will include the full repertoire of growth factors from the perihepatic mesenchyme and the first detailed knowledge of native human hepatoblasts, early hepatocytes, ductal plate cells and cholangiocytes. We will do this by single cell RNA-seq and computational biology and expect to discover distinct cell sub-populations currently unaccounted for in PSC differentiation protocols. Based on previous experience, we expect the data to lead us to mechanistic understanding and critical transcription factors for reprogramming. By the end of the project, we will have discovered and disseminated the first molecular basis for lineage differentiation within the human embryonic liver. We will have pilot-tested some of the emergent key factors in our comprehensive platforms of PSC differentiation and cell reprogramming. Together, we hope our freely available knowledge will act as a watershed that unlocks major improvements in PSC-to-liver and hepatic reprogramming research amongst the international community.

Our work has major impact as evidenced by the journals in which our research is published, their impact factors and our citation rates. Very few groups devote themselves to post-implantation human developmental biology and allied aspects of stem cell biology. The PI made this choice 20 years ago as where his group could make its biggest contribution to the scientific community. Our data directly underpin human stem cell biology, e.g. by removing the need for inter-species extrapolation. As we realise that important nuances of cell phenotype frequently involve species-specific attributes, and as we ultimately aspire to human clinical therapy grade cells, this knowledge base directly in developing human cell-types is critically important.

Directly related to this project:

1. We will provide the first in depth molecular phenotype of the key liver cell-types via cutting-edge technology. Without our work, these genome-wide data are unattainable by the vast majority of researchers. Following dissemination of our work the data can be reapplied by other scientists in their projects. Next-to-nothing is known about the molecular phenotype of these native human embryonic cells currently. It is highly likely that we will discover new stages of human liver cell differentiation that are currently unrepresented in PSC differentiation protocols.

2. We will provide the first mechanistic detail on how the human liver develops.

3. We will make freely available prioritised factors to improve stem cell-to-liver differentiation and cellular reprogramming. Currently, stem cell protocols derive almost entirely from knowledge of mouse development while reprogramming choices rely on user-defined decisions or information from fully differentiated cell-types.

4. Our datasets will be freely available for download via our open access website.