Research Article: Production of Factor VIII by Human Liver Sinusoidal Endothelial Cells Transplanted in Immunodeficient uPA Mice

Date Published: October 22, 2013

Publisher: Public Library of Science

Author(s): Marina E. Fomin, Yanchen Zhou, Ashley I. Beyer, Jean Publicover, Jody L. Baron, Marcus O. Muench, Peter J. Lenting.

http://doi.org/10.1371/journal.pone.0077255

Abstract

Liver sinusoidal endothelial cells (LSECs) form a semi-permeable barrier between parenchymal hepatocytes and the blood. LSECs participate in liver metabolism, clearance of pathological agents, immunological responses, architectural maintenance of the liver and synthesis of growth factors and cytokines. LSECs also play an important role in coagulation through the synthesis of Factor VIII (FVIII). Herein, we phenotypically define human LSECs isolated from fetal liver using flow cytometry and immunofluorescence microscopy. Isolated LSECs were cultured and shown to express endothelial markers and markers specific for the LSEC lineage. LSECs were also shown to engraft the liver when human fetal liver cells were transplanted into immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene (uPA-NOG mice). Engrafted cells expressed human Factor VIII at levels approaching those found in human plasma. We also demonstrate engraftment of adult LSECs, as well as hepatocytes, transplanted into uPA-NOG mice. We propose that overexpression of uPA provides beneficial conditions for LSEC engraftment due to elevated expression of the angiogenic cytokine, vascular endothelial growth factor. This work provides a detailed characterization of human midgestation LSECs, thereby providing the means for their purification and culture based on their expression of CD14 and CD32 as well as a lack of CD45 expression. The uPA-NOG mouse is shown to be a permissive host for human LSECs and adult hepatocytes, but not fetal hepatoblasts. Thus, these mice provide a useful model system to study these cell types in vivo. Demonstration of human FVIII production by transplanted LSECs encourages further pursuit of LSEC transplantation as a cellular therapy for the treatment of hemophilia A.

Partial Text

Liver sinusoidal endothelial cells (LSECs) form a layer partially separating hepatocytes from the blood in liver sinusoids. LSECs play a number of important roles including blood filtration, cytokine secretion and immune regulation [1]–[3]. LSECs differ from vascular endothelial cells in that they lack a basal membrane and have large pores called fenestrae. Fenestrae allow passage of plasma and particles between blood and the space of Disse, thus allowing hepatocytes to perform their metabolic functions [4]. LSECs originate during embryogenesis from the capillary endothelium of the septum transversum [5]. Between 5 and 20 weeks’ gestation LSECs lose their basal membranes, acquire fenestrations and start production of extracellular matrix components.

Successful transplantation of human liver cells into uPA-NOG mice was demonstrated resulting in engraftment of LSECs and production of human FVIII. We present an extensive phenotypic profile of fetal LSECs, which should prove valuable in future efforts in the identification, isolation and study of LSECs. Freshly isolated and transplanted LSECs can be reliably identified based on their expression of CD14 and CD32 as well as a lack of CD45 staining to exclude hematopoietic cells such as macrophages. Furthermore, we demonstrated that whereas fetal and adult LSECs engrafted uPA-NOG mice, only adult hepatocytes could engraft uPA-NOG mice.

 

Source:

http://doi.org/10.1371/journal.pone.0077255