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

<div><p>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.</p></div

Transplantation of adult human liver cells results in hepatocyte and LSEC engraftment.

<p>Examples of hepatocytes are shown in (<b>A</b>) and LSECs in (<b>B</b>). Mouse H-2K^d is shown in red; human markers are shown in green. Nuclei are stained with DAPI (blue).</p

Gene expression by fetal LSECs. CD31 expression on CD14⁺⁺ cells measured by flow cytometry and mRNA levels.

<p>(<b>A</b>). Viable non-hematopoietic cells (PI⁻CD45⁻) gated by CD326 and CD14 expression (a: CD14⁻CD326⁻; b: CD326^loCD14⁻; c: CD14⁺⁺CD326^+/−; d: CD326⁺⁺CD14^lo). Expression of CD31 was analyzed by qPCR in each sorted population (n = 3). Flow cytometric plots shows expression of CD31 versus CD326 and CD31 versus CD14. (<b>B</b>): Gene expression profile of sorted populations (a: CD326^loCD14⁻; b: CD14⁺⁺CD326⁻; c: CD14⁺⁺CD326⁺; d: CD326⁺⁺CD14^lo) measured by qPCR, n = 7.</p

Analysis of human fetal liver cell engraftment in mouse livers.

<p>Flow cytometric analysis is shown comparing livers from untransplanted and transplanted mice. Live cells were stained with mouse markers CD45, TER-119 and H-2K^d and human B2M. CD14 and CD45 expression define two populations of B2M⁺ cells (<b>A</b>). A summary of 6 experiments used 44 transplanted mice shows the ratio of CD45⁻ cells among all human cells engrafted. The trend line shows a tendency of total, B2M⁺ cell, engraftment to increase with time whereas the CD45⁻ population remains more constant (<b>B</b>). Analysis of endothelial cell markers expression on the light-density fraction of transplanted mouse liver cells. Expression of the indicated antigens on CD14⁺⁺CD45⁻ cells is shown with filled histograms and staining with isotype controls are shown as unfilled histogram (<b>C</b>).</p

Transplanted human cells produce FVIII.

<p>The graph represents ELISA measurements of human FVIII in the plasma of untransplanted uPA-NOG mice (n = 5) and mice transplanted with human fetal liver cells (n = 22). Results are compared to a calibrated human plasma standard from the assay manufacturer and an independent human plasma sample obtained from our institute (n = 3). The calibrated plasma standard has 108% FVIII activity of a reference standard, which is equivalent to 0.95 IU/ml. Data are shown as the mean ± standard deviation.</p

In vivo gene expression by fetal LSECs revealed by immunofluorescence staining.

<p>Fetal livers of age 9.5 weeks (A, B), 24 weeks (C–L), and 19 weeks (M–R) stained with endothelial markers. Hepatocytes stained with CD326 (A–C), nuclei stained by DAPI (blue, A–F, I, L–R). I is merged from G and H, L is merged from J and K. s-sinusoid, v-vessel with vascular endothelium.</p

CD45 expression distinguishes LSECs from liver macrophages (MØ).

<p>Multiple populations of CD14⁺⁺ cells are distinguished by CD45 expression (<b>A</b>). LSECs represent a population of CD14⁺⁺ cells found among CD45⁻ cells, whereas MØ express high levels of CD45 as indicated by the oval region. Antigen expression is compared on MØ and LSECs (<b>B</b>). Background staining with isotype-control antibody is shown for the oval MØ region. The corresponding antigen expression found on LSECs is shown by gating on CD14⁺⁺CD45⁻ cells as defined in Fig. 3.</p

Mouse VEGF expression in livers (A) and plasma (B) of uPA-NOG homozygous, uPA-NOG hemizygous and NSG mice (n = 9 for each group).

<p>VEGF levels in the liver are shown as ratio of the amount of VEGF to total protein.</p

Flow cytometric analysis of antigens on LSECs.

<p>LSECs were defined as CD14⁺⁺CD45⁻ cells using a CD45⁻ gate as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0077255#pone-0077255-g004" target="_blank">Fig 4A</a> and a CD14⁺⁺ gate as indicated by region c in Fig. 1A. Expression of the indicated antigens is shown using filled histograms, whereas staining with the corresponding isotype-control antibody is shown using an unfilled histogram. The mean frequency of positive events and the number (n) of specimens analyzed are shown on the right.</p

Summary of cell and recipient uPA-NOG mouse numbers used in individual transplant experiments.

*<p>The actual amount of cells transplanted into each animal can in some cases be lower because of the sample leakage during injections.</p