Table 1.
Summary of cell and recipient uPA-NOG mouse numbers used in individual transplant experiments.
Figure 1.
Gene expression by fetal LSECs. CD31 expression on CD14++ cells measured by flow cytometry and mRNA levels.
(A). Viable non-hematopoietic cells (PI−CD45−) gated by CD326 and CD14 expression (a: CD14−CD326−; b: CD326loCD14−; c: CD14++CD326+/−; d: CD326++CD14lo). 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): Gene expression profile of sorted populations (a: CD326loCD14−; b: CD14++CD326−; c: CD14++CD326+; d: CD326++CD14lo) measured by qPCR, n = 7.
Figure 2.
In vivo gene expression by fetal LSECs revealed by immunofluorescence staining.
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.
Figure 3.
Flow cytometric analysis of antigens on LSECs.
LSECs were defined as CD14++CD45− cells using a CD45− gate as shown in Fig 4A 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.
Figure 4.
CD45 expression distinguishes LSECs from liver macrophages (MØ).
Multiple populations of CD14++ cells are distinguished by CD45 expression (A). 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). 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.
Figure 5.
Morphology and antigen expression on cultured CD14++CD326−CD45− cells.
Cultured cells form a “cobble stone” monolayer and express the indicated endothelial markers (red). Nuclei stained by DAPI (blue). 200×.
Figure 6.
Analysis of human fetal liver cell engraftment in mouse livers.
Flow cytometric analysis is shown comparing livers from untransplanted and transplanted mice. Live cells were stained with mouse markers CD45, TER-119 and H-2Kd and human B2M. CD14 and CD45 expression define two populations of B2M+ cells (A). 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). 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 (C).
Figure 7.
Culture of human cells recovered from the livers of transplanted uPA-NOG mice.
Livers were enzymatically digested and light density cells isolated, which were cultured on collagen-coated plates with EBM2 supplied medium. Cells became confluent after 3 weeks, forming a cobble-stone layer typical for cultured human endothelial cells. The cells expressed human CD31 (green) and were negative for staining with mouse H-2Kd (not shown). Nuclei are stained with DAPI (blue).
Figure 8.
Transplanted human fetal liver cells engrafted in mouse liver.
Human B2M (green) stains small elongated cells lining sinusoids between the larger mouse hepatocytes in mice transplanted with fetal human liver. Mouse cells are stained by H-2Kd (red). Human hematopoietic B2M+ cells are seen in a close-up view of a vessel. Human LSEC markers CD14, CD31, CD32, CD32b, CD34 and CD105 (green) stain small elongated cells located between mouse hepatocytes and inside sinusoids. Nuclei stained with DAPI are shown in blue.
Figure 9.
Transplanted human cells produce FVIII.
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.
Figure 10.
Mouse VEGF expression in livers (A) and plasma (B) of uPA-NOG homozygous, uPA-NOG hemizygous and NSG mice (n = 9 for each group).
VEGF levels in the liver are shown as ratio of the amount of VEGF to total protein.
Figure 11.
Transplantation of adult human liver cells results in hepatocyte and LSEC engraftment.
Examples of hepatocytes are shown in (A) and LSECs in (B). Mouse H-2Kd is shown in red; human markers are shown in green. Nuclei are stained with DAPI (blue).