Human Liver Endothelial Cells, But Not Macrovascular or Microvascular Endothelial Cells, Engraft in the Mouse Liver

Abstract

Liver cell transplantation has had limited clinical success so far, partly due to poor engraftment of hepatocytes. Instead of hepatocytes. other cell types, such as endothelial cells, could be used in ex vivo liver gene therapy. The goal of the present study was to compare the grafting and repopulation capacity of human endothelial cells derived from various tissues. Human endothelial cells were isolated from adult and fetal livers using anti-human CD31 antibody-conjugated magnetic beads. Human macrovascular endothelial cells were obtained from umbilical vein. Human microvascular endothelial cells were isolated from adipose tissue. Cells were characterized using flow cytometry. Liver engraftment and repopulation of endothelial cells was studied after intrasplenic transplantation in monocrotaline-treated immunodeficient mice. Following transplantation, human liver endothelial cells engrafted throughout the mouse liver. With immunoscanning electron microscopy, fenestrae in engrafted human liver endothelial cells were identified, a characteristic feature of liver sinusoidal endothelial cells. In contrast, CD31-negative liver cells, human macrovascular and microvascular endothelial cells were not capable of repopulating mouse liver. Characterization of human liver, macrovascular, and microvascular endothelial cells demonstrated expression of CD31, CD34, and CD146 but not CD45. Our study shows that only human liver endothelial cells, but not macro-and microvascular endothelial cells, have the unique capacity to engraft and repopulate the mouse liver. These results indicate that mature endothelial cells cannot transdifferentiate in vivo and thus do not exhibit phenotypic plasticity. Our results have set a basis for further research to the potential of human liver endothelial cells in liver-directed cell and gene therapy.

Hepatitis C Virus Infection Induces Autocrine Interferon Signaling by Human Liver Endothelial Cells and Release of Exosomes, Which Inhibits Viral Replication

“…We used isolated primary liver endothelial cells described recently 17 , ex vivo purified LSECs, as well as an immortalized HLSEC line, TMNK-1 205556 . Prior reports indicate that CD31 + LYVE-1 + liver endothelial cells lose their fenestrae in culture 57 , but nonetheless, when transplanted into mice, exhibit fenestrae in vivo , suggesting that in the appropriate liver microenvironment, these cells resemble true LESCs 45 . We considered using VEGF-A in order to induce fenestrations 5859 , but decided against that approach because it could potentially complicate analyses on HCV replication 1560 .…”

“…Despite the fact LSECs are the first cells in contact with blood flow in hepatic sinusoids and account for the largest proportion of non-parenchymal cells in the liver 45 , little is known about how these cells recognize hepatitis C. Given their strategic anatomic location 46 we reasoned that human LSECs (HLSECs) could play a central antiviral role. We show that HLSECs express many of the requisite receptors for antiviral recognition, including TLRs and retinoic acid inducible gene-I (RIG-I), the cytoplasmic sensor of HCV.…”

Specific gene delivery to liver sinusoidal and artery endothelial cells

“…35 Resting vascular ECs express only low levels of CD105 and, moreover, the velocity of blood flow is relatively high in large vessels, but slows down substantially in the sinuses of the liver. 3,18 At low flow rates the likelihood for functional contacts between CD105-LV and CD105 leading to gene delivery is considerably enhanced. This explains the efficient gene delivery observed for mCD105-LV and for huCD105-LV in EC-transplanted mice.…”

“…Mice were sacrificed and fixed by in vivo perfusion, and livers were excised for histology and immunohistochemistry as described. 18 After erythropoietin gene transfer, hematology was performed on a Scil Vet ABCCounter (Scil Animal Care, Viernheim, Germany).…”

“…18 Only CD31-positive cells exhibiting the typical morphology of ECs were transduced by huCD105-LV ( Figure 5A). No murine LSECs or F4/ 80-positive Kupffer cells were transduced, demonstrating the cell specificity of huCD105-LV ( Figure 5A).…”