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.

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.

Publications

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Adams DH (2018) Could endothelial TGFß signaling be a promising new target for liver disease? in Expert review of gastroenterology & hepatology

 
Description Scientific Advisory Board for Stem Cells For Safer Medicines
Geographic Reach National 
Policy Influence Type Participation in advisory committee
Impact Use of stem cell derived hepatocytes for drug testing is the remit of Stem cells for Safer medicines. I advised them on their scientific board. This has the potential to improve drug testing and accelerate new therapies.
 
Description Birmingham Children's Hospital Charitable Funds
Amount £73,000 (GBP)
Organisation BCH Charities 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2010 
End 11/2011
 
Title Establishing human ES culture in Birmingham 
Description Its establishemnt here has allowed other groups to use the technology for their own research ideas/work. 
Type Of Material Improvements to research infrastructure 
Year Produced 2010 
Provided To Others? Yes  
Impact None yet 
 
Description Collaboration to develop iPS cells 
Organisation University of Birmingham
Department College of Medical and Dental Sciences
Country United Kingdom 
Sector Academic/University 
PI Contribution We were approached in view of the funding and expereince in the area.
Collaborator Contribution It has devleoped the scope and application of the original project
Impact No publications yet, although the work is progressing well.
Start Year 2009
 
Description Collaboration to study engraftment of ES derived hepatocytes 
Organisation University of Manchester
Country United Kingdom 
Sector Academic/University 
PI Contribution We instigated the possibility of them working with us to test the efficacy of their cells
Collaborator Contribution Will allow us to compare the efficacy of different populations of cells
Impact None yet
Start Year 2011
 
Title Differentiation protocol 
Description I was involved in the generation of a differentiation protocol for making functional hepatocytes from human ES cells. Thi shas beenhighly cited and is used by many industrial companies. 
Type Therapeutic Intervention - Cellular and gene therapies
Current Stage Of Development Refinement. Non-clinical
Year Development Stage Completed 2010
Development Status Under active development/distribution
Impact Improved efficency of making hES derived hepatocytes. This will be important for drug testing. 
 
Description British Science Festival 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Primary Audience Public/other audiences
Results and Impact General public attended for a debate/discussion on role of embryonic stem cells in patient care.

Request from audeince for more information about research
Year(s) Of Engagement Activity 2010
 
Description Stem cell debate 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Type Of Presentation Keynote/Invited Speaker
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Debate about the role and ethics of stem cell therapies.

I have been asked to come and speak at school days
Year(s) Of Engagement Activity 2012
 
Description UNiversity open day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Public/other audiences
Results and Impact 40 members of the public heard about the impact stem cells could have in clinical medicine

Questioning of real potential of stem cell therapy.
Year(s) Of Engagement Activity 2011