Pre-clinical study of hepatocyte transplantation using human embryonic stem cell-derived hepatocytes
Lead Research Organisation:
University of Birmingham
Department Name: Immunity and Infection
Abstract
In the UK liver disease is the fifth most common cause of death, and it is rising dramatically. Whole organ liver transplantation is limited in its availability, it is not capable of meeting the clinical need. Other options include infusing suspensions of liver cells into patients with liver disease, but this is again limited by a lack of livers. Thus, there is a clear need to expand functional liver cells for clinical applications such as liver cell transplantation. In collaboration with Geron, we have generated highly efficient methods to direct human embryonic stem cells down the liver cell lineage resulting in 90% purity.
There are two main issues to be overcome before these cells can be used clinically.
(1) The delivery of these cells within the liver needs to be improved as at present we can only deliver small numbers of cells. (2) There are concerns that cells derived from embryonic stem cells can form tumours after transplantation.
We will identify the molecules which control the entry of infused liver cells into the liver, and augment their activity, thus improving the number of cells entering. Initially we will do this with liver cells in a complex multi-cellular culture model which mimics what happens in a living organ. Having identified which molecules are most important in improving the engraftment of liver cells in tissue culture we will then proceed to a mouse model where we can test the effects of stimulating these molecules in a living organism. Of note we will use a mouse which has a metabolic liver disorder, which means that as well as being able to quantify the number of human liver cells which have engrafted within the mouse liver we can also see what impact they have had on the metabolic defect. This is timely, as it is likely that the first clinical studies with such cells will occur in patients with metabolic liver disease.
By purifying the population of liver cells we infuse as well as limiting the route of administration we intend to demonstrate that we can completely abrogate the risk of tumour development. There is however a theoretical risk that transplanting human embryonic stem liver cells into a mouse will not adequately assess the risk of tumour development. As a fail-safe therefore we will also perform experiments in which we infuse mouse embryonic stem liver cells into the mouse to exclude this possibility.
There are two main issues to be overcome before these cells can be used clinically.
(1) The delivery of these cells within the liver needs to be improved as at present we can only deliver small numbers of cells. (2) There are concerns that cells derived from embryonic stem cells can form tumours after transplantation.
We will identify the molecules which control the entry of infused liver cells into the liver, and augment their activity, thus improving the number of cells entering. Initially we will do this with liver cells in a complex multi-cellular culture model which mimics what happens in a living organ. Having identified which molecules are most important in improving the engraftment of liver cells in tissue culture we will then proceed to a mouse model where we can test the effects of stimulating these molecules in a living organism. Of note we will use a mouse which has a metabolic liver disorder, which means that as well as being able to quantify the number of human liver cells which have engrafted within the mouse liver we can also see what impact they have had on the metabolic defect. This is timely, as it is likely that the first clinical studies with such cells will occur in patients with metabolic liver disease.
By purifying the population of liver cells we infuse as well as limiting the route of administration we intend to demonstrate that we can completely abrogate the risk of tumour development. There is however a theoretical risk that transplanting human embryonic stem liver cells into a mouse will not adequately assess the risk of tumour development. As a fail-safe therefore we will also perform experiments in which we infuse mouse embryonic stem liver cells into the mouse to exclude this possibility.
Technical Summary
Chronic liver disease is an increasing cause of morbidity & mortality in the UK. Treatments such as hepatocyte transplantation are limited by a lack of hepatocytes and poor engraftment in the recipient liver. We can efficiently differentiate human embryonic stem (hES) cells down the hepatocytic lineage, and demonstrate high levels of metabolic function in vitro and in vivo after transplanting these cells into immunocompromised mice. There remains a concern that hES-derived cells may generate teratomas in vivo.
In this proposal we address the two major hurdles to the clinical translation of hES-derived hepatocyte transplantation, which are (i) improving the engraftment of hepatocytes into the liver, and (ii) abrogating the risk of teratoma development in vivo.
(i) Improving engraftment. Using an established co-culture trans-migration assay to model hepatocyte transplantation in vitro, we will define the role of candidate receptors/molecules (?O1-integrin, G-protein coupled receptors, CD44 and vEGF) in this process & develop clinically applicable manipulations to improve the efficiency of this process. We will test the impact of relevant receptor manipulations in vivo using the Fah-/-/Rag2-/-/Il2r??-/- (FRG) mouse which develops tyrosinaemia due to a metabolic defect. Using this model we can quantify the number of engrafted hESHs and their correction of the metabolic defect. This is clinically relevant, as we predict metabolic liver disease will be the likely first clinical application of stem cell therapy.
(ii) To implement procedures to reduce teratoma risk in vivo we will examine:
(a) the effects of cell purification before transplantation, by only infusing hESH cells (by MACS sorting) which express mature hepatocyte markers (asialoglycoprotein receptor). We predict that infusions containing poorly/undifferentiated cells will have a higher teratoma risk.
(b) the route of cell administration, by comparing systemic (intra-splenic) infusions of hESH cells with local (intra-portal) infusions into the FRG mice. We predict that local infusions will have a lower teratoma risk.
(c) the utility of xeno-transplants in assessing teratoma risk. There is a concern that xeno-transplants in immunocompromised mice may under-estimate the teratoma risk due to species differences. We will transplant murine ESHs into the FRG mouse to see if this results in an increase in teratoma risk.
At completion of this project we will have addressed the two major hurdles to the clinical translation of (human ES derived) hepatocyte transplantation, and be positioned to move into preliminary clinical studies.
In this proposal we address the two major hurdles to the clinical translation of hES-derived hepatocyte transplantation, which are (i) improving the engraftment of hepatocytes into the liver, and (ii) abrogating the risk of teratoma development in vivo.
(i) Improving engraftment. Using an established co-culture trans-migration assay to model hepatocyte transplantation in vitro, we will define the role of candidate receptors/molecules (?O1-integrin, G-protein coupled receptors, CD44 and vEGF) in this process & develop clinically applicable manipulations to improve the efficiency of this process. We will test the impact of relevant receptor manipulations in vivo using the Fah-/-/Rag2-/-/Il2r??-/- (FRG) mouse which develops tyrosinaemia due to a metabolic defect. Using this model we can quantify the number of engrafted hESHs and their correction of the metabolic defect. This is clinically relevant, as we predict metabolic liver disease will be the likely first clinical application of stem cell therapy.
(ii) To implement procedures to reduce teratoma risk in vivo we will examine:
(a) the effects of cell purification before transplantation, by only infusing hESH cells (by MACS sorting) which express mature hepatocyte markers (asialoglycoprotein receptor). We predict that infusions containing poorly/undifferentiated cells will have a higher teratoma risk.
(b) the route of cell administration, by comparing systemic (intra-splenic) infusions of hESH cells with local (intra-portal) infusions into the FRG mice. We predict that local infusions will have a lower teratoma risk.
(c) the utility of xeno-transplants in assessing teratoma risk. There is a concern that xeno-transplants in immunocompromised mice may under-estimate the teratoma risk due to species differences. We will transplant murine ESHs into the FRG mouse to see if this results in an increase in teratoma risk.
At completion of this project we will have addressed the two major hurdles to the clinical translation of (human ES derived) hepatocyte transplantation, and be positioned to move into preliminary clinical studies.