49 The Bipotential Human EpCAM+CD24+CD133+Cholangiocyte is a Transplantable Regenerative Therapy for Biliary Disese
John Hallett1,2, Wei-Yu Lu1, Sofia Ferreira-Gonzalez1, Tak Yung Man1, John Thomson1, Eoghan O'Duibbhir3, Victoria Gadd1, Dwyer Benjamin1, Kayleigh Thirlwell4, Alasdair Fraser4, David Hay1, Gabriel Oniscu5,1, Stuart Forbes1,5
1University of Edinburgh, Edinburgh, United Kingdom. 2Royal Hospital for Sick Children Edinburgh, Edinburgh, United Kingdom. 3University of Edinburgh, EdinburghE, United Kingdom. 4Scottish National Blood Transfusion Service, Edinburgh, United Kingdom. 5Royal Infirmary of Edinburgh, Edinburgh, United Kingdom

Abstract

Aim
Biliary disorders are rare but serious causes of childhood morbidity and mortality. We propose that bipotential liver progenitor cells can be isolated from human livers, expanded in vitro and used as a transplantable therapy for biliary disease.
Methods
Human livers discarded from the transplantation process were digested and cell populations isolated, cultured and analysed using RNA Sequencing, flow cytometry, immunohistochemistry and functional analysis. These cells were then transplanted into the Krt19CreERMdm2fl/flRag2-/-Il2rg-/- mouse, a novel model of biliary disease. Biochemistry, immunohistochemistry, histology and qPCR were performed with appropriate statistics.
Results
Human livers were digested and EpCAM+CD24+CD133+ cells were isolated and expanded as organoids. These cells made up 1% of the total cholangiocyte yield. They had a superior single cell colony forming ability compared to EpCAM+CD24+CD133- cells (1.67 relative increase p<0.01) RNA Sequencing from multiple donors revealed the cells were positive for genes associated with multipotency (Sox9, FoxA2). EpCAM+CD24+CD133+ cells were maintained in culture 1 year (whilst EpCAM+CD24+CD133cells did not survive beyond 15 weeks. They were differentiated towards a hepatocyte phenotype (positive staining for CYP2D6  and albumin and negative for CK19) while controls maintained a biliary phenotype.
When transplanted into the Krt19CreERMdm2fl/flRag2-/-Il2rg-/- model of biliary disease all animals treated survived well to day 42 with normalising liver biochemistry and histology. Controls had deranged liver biochemistry, necrotic and inflamed livers and reached clinical endpoints for euthanization. Human cells were found engrafted in the bile ducts of the treated mice.
Conclusion
A bipotential human hepatic progenitor cell can be isolated from discarded transplant livers, expanded in culture and be transplanted into a model of biliary disease, resulting in engraftment and recovery from biliary injury indicating a potential future cell therapy.


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