Immortalized porcine liver sinusoidal endothelial cells: an in vitro model of xenotransplantation-induced thrombocytopenia.
BACKGROUND
Xenotransplantation has the potential to solve the critical shortage of human organs available for allotransplantation. The major barrier to porcine liver xenotransplantation is sequestration of human platelets causing thrombocytopenia. Porcine liver sinusoidal endothelial cells (LSEC) bind and phagocytose human platelets at least in part through binding of the asialoglycoprotein receptor 1 (ASGR1). Our purpose was to generate an immortalized porcine LSEC (iLSEC) line that mimics primary LSEC in ASGR1 expression and phagocytosis of human platelets. Porcine iLSEC would enable continued study of xenotransplantation-induced thrombocytopenia in vitro with fewer animals sacrificed.


METHODS
Primary domestic porcine LSEC were transduced with lentiviral vector expressing the large and small T antigen of SV40 (SV40 TAg). The phenotype and genotype of the immortalized LSEC were compared with primary LSEC.


RESULTS
A total of eight clones expressing SV40 TAg were isolated, and one clone was subcultured and analyzed for growth, phenotype, and function during passages 15-40. Expression of the SV40 TAg was confirmed by confocal microscopy and western blot. MTS cell proliferation assay demonstrated that the clone rapidly grew in culture medium with 2-10% fetal bovine serum. iLSEC expressed the endothelial cell marker, CD31, as determined by confocal microscopy and flow cytometry. Activation of iLSEC by treatment with lipopolysaccharide (LPS) resulted in upregulation of the inflammatory cytokine interleukin 6 (IL 6) by qPCR and ELISA. iLSEC phagocytosed human serum albumin and latex beads as measured by flow cytometry. Human platelets were phagocytosed by immortalized porcine LSEC.


CONCLUSIONS
Immortalized porcine LSEC retain a phagocytic phenotype, making them a good model for the study of xenotransplantation-induced thrombocytopenia and may provide further insight into the phagocytic role of LSEC.
Department of Surgery, Indiana University School of Medicine, Indianapolis, IN, USA.
Zheng-Yu
ZY
Wang
Leela L
LL
Paris
Ray K
RK
Chihara
A Joseph
AJ
Tector
Christopher
C
Burlak
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics; D037263:Asialoglycoprotein Receptor / Q000378:metabolism; D001792:Blood Platelets / Q000378:metabolism; D002417:Cattle; D002461:Cell Line, Transformed; D049109:Cell Proliferation; D042783:Endothelial Cells / Q000502:physiology / Q000637:transplantation; D022781:Hepatocytes / Q000502:physiology / Q000637:transplantation*; D006801:Humans; D066298:In Vitro Techniques; D015850:Interleukin-6 / Q000378:metabolism; D008099:Liver / Q000166:cytology; D008954:Models, Biological; D010587:Phagocytosis; D034421:Sus scrofa; D013552:Swine; D013921:Thrombocytopenia / Q000209:etiology*; D014183:Transplantation, Heterologous / Q000009:adverse effects*
D000952:Antigens, Polyomavirus Transforming; D037263:Asialoglycoprotein Receptor; D015850:Interleukin-6