Liver specification of human iPSC-derived endothelial cells transplanted into mouse liver
Kiryu K. Yap,
Jan Schröder,
Yi-Wen Gerrand,
Aleksandar Dobric,
Anne M. Kong,
Adrian M. Fox,
Brett Knowles,
Simon W. Banting,
Andrew G. Elefanty,
Eduoard G. Stanley,
George C. Yeoh,
Glen P. Lockwood,
Victoria C. Cogger,
Wayne A. Morrison,
Jose M. Polo,
Geraldine M. Mitchell
Affiliations
Kiryu K. Yap
O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia; University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia; Corresponding author. Address: St Vincent’s Institute. 9 Princes Street, Fitzroy, VIC, 3065 Australia. Tel.: +613 9231 2480; Fax: +613 9416 2676.
Jan Schröder
Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia; Australian Regenerative Medicine Institute, Clayton, VIC, Australia; Doherty Institute & University of Melbourne Department of Microbiology and Immunology, Parkville, VIC, Australia
Yi-Wen Gerrand
O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
Aleksandar Dobric
O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
Anne M. Kong
O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia
Adrian M. Fox
University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia; Hepatobiliary Surgery Unit, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
Brett Knowles
University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia; Hepatobiliary Surgery Unit, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
Simon W. Banting
University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia; Hepatobiliary Surgery Unit, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia
Andrew G. Elefanty
Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
Eduoard G. Stanley
Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Murdoch Children's Research Institute, The Royal Children's Hospital, Flemington Road, Parkville, VIC, Australia; Department of Paediatrics, Faculty of Medicine, Dentistry and Health Sciences, University of Melbourne, Parkville, VIC, Australia
George C. Yeoh
Harry Perkins Institute of Medical Research and Centre for Medical Research, University of Western Australia, Perth, WA, Australia
Glen P. Lockwood
ANZAC Research Institute and University of Sydney, Concord, NSW, Australia
Victoria C. Cogger
ANZAC Research Institute and University of Sydney, Concord, NSW, Australia
Wayne A. Morrison
O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia; University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia; Australian Catholic University, Fitzroy, VIC, Australia
Jose M. Polo
Department of Anatomy and Developmental Biology, Monash University, Clayton, VIC, Australia; Monash Biomedicine Discovery Institute, Monash University, Clayton, VIC, Australia; Australian Regenerative Medicine Institute, Clayton, VIC, Australia; Adelaide Centre for Epigenetics, South Australian Immunogenomics Cancer Institute, University of Adelaide, Adelaide, SA, Australia
Geraldine M. Mitchell
O’Brien Department of St Vincent’s Institute, Fitzroy, VIC, Australia; University of Melbourne Department of Surgery, St Vincent’s Hospital Melbourne, Fitzroy, VIC, Australia; Australian Catholic University, Fitzroy, VIC, Australia
Background & Aims: Liver sinusoidal endothelial cells (LSECs) are important in liver development, regeneration, and pathophysiology, but the differentiation process underlying their tissue-specific phenotype is poorly understood and difficult to study because primary human cells are scarce. The aim of this study was to use human induced pluripotent stem cell (hiPSC)-derived LSEC-like cells to investigate the differentiation process of LSECs. Methods: hiPSC-derived endothelial cells (iECs) were transplanted into the livers of Fah−/−/Rag2−/−/Il2rg−/− mice and assessed over a 12-week period. Lineage tracing, immunofluorescence, flow cytometry, plasma human factor VIII measurement, and bulk and single cell transcriptomic analysis were used to assess the molecular and functional changes that occurred following transplantation. Results: Progressive and long-term repopulation of the liver vasculature occurred as iECs expanded along the sinusoids between hepatocytes and increasingly produced human factor VIII, indicating differentiation into LSEC-like cells. To chart the developmental profile associated with LSEC specification, the bulk transcriptomes of transplanted cells between 1 and 12 weeks after transplantation were compared against primary human adult LSECs. This demonstrated a chronological increase in LSEC markers, LSEC differentiation pathways, and zonation. Bulk transcriptome analysis suggested that the transcription factors NOTCH1, GATA4, and FOS have a central role in LSEC specification, interacting with a network of 27 transcription factors. Novel markers associated with this process included EMCN and CLEC14A. Additionally, single cell transcriptomic analysis demonstrated that transplanted iECs at 4 weeks contained zonal subpopulations with a region-specific phenotype. Conclusions: Collectively, this study confirms that hiPSCs can adopt LSEC-like features and provides insight into LSEC specification. This humanised xenograft system can be applied to further interrogate LSEC developmental biology and pathophysiology, bypassing current logistical obstacles associated with primary human LSECs. Impact and implications: Liver sinusoidal endothelial cells (LSECs) are important cells for liver biology, but better model systems are required to study them. We present a pluripotent stem cell xenografting model that produces human LSEC-like cells. A detailed and longitudinal transcriptomic analysis of the development of LSEC-like cells is included, which will guide future studies to interrogate LSEC biology and produce LSEC-like cells that could be used for regenerative medicine.
