Figure 1.
Engraftment of the murine liver with human blood cells.
(A) A NOD-SCID mouse transplanted with human CD34++CD45+ hFL cells was analyzed 71 days after transplant revealing high level engraftment of human CD45+CD59+ cells in the BM, spleen and liver; no clear indication of circulating human cells was seen in the blood. (B) Box plot showing the median and range of human leukocyte engraftment in 4 transplanted NOD-SCID mice from 2 independent experiments analyzed at 67 and 71 days post transplant. Significant differences among all possible pair-wise comparisons are indicated.
Figure 2.
Mature human blood cells in the murine liver.
(A) Significantly more light-density liver cells were recovered from 3 mice transplanted with CD34++CD38− hFL cells than from untransplanted NSG mice. (B) Myeloid, lymphoid and erythroid engraftment observed 68-166 days after transplantation with hFBM or Lin− LDFL cells. (C) Distribution of T-cell subsets among CD3+ T-cells in mice transplanted with hFBM cells. The numbers (n) of animals evaluated are indicate in the 3 box plots. (D) Flow cytometric analysis of light-density liver cells pooled from 3 NSG mice transplanted with CD34++CD38− hFL cells were analyzed 144 days after transplantation showing multilineage hematopoietic engraftment. T-cell subsets were evaluated by gating on CD3+ cells as indicated. CD56+ NK cells were defined by a low side-light scatter gate (not shown) and their lack of CD3 expression. Numbers shown in the graphs represent the percentages of gated events among all CD59+ human cells.
Figure 3.
Mast cells are present in the liver of chimeric mice and in human prenatal development.
(A) Light-density liver cells, pooled from 9 chimeric mice, were analyzed 89 days after transplantation with hFBM cells. CD117++CD203c+ mast cells are indicated by the rectangular gate, which also express low levels of CD45. (B) CD117++CD203c+ mast cells were found in human hFBM at 20 weeks' gestation, which also express low levels of CD34 and CD45. (C) CD117++CD203c+ mast cells were observed in hFL at 18 weeks' gestation. Antigen expression on CD117++ mast cells, gated as indicated in the dot plot, are shown using histograms. The frequency of positive cells, relative to isotype controls shown in grey outline, are indicated in each plot.
Figure 4.
Human hematopoiesis in the livers of mice transplanted with fetal cells.
(A) Hematopoietic precursors are present in the liver of a mouse analyzed 130 days after being transplanted with 1×106 hFL cells. Filled arrows identify CD38−CD34++ and CD133+CD34++ cells, possible HSCs, whereas open arrows point to committed progenitors. (B) Various committed hematopoietic progenitor populations are evident in the liver including B-cell progenitors and myeloid progenitors shown from among CD19− human cells.The bottom row of data shows immature erythroid cells that express low levels of CD235a and high levels of CD71 as indicated by the arrows. Data are from 4 pooled livers analyzed 148 days after transplantation with 2×105 Lin− hFL cells. (C) HPP-CFC and LPP-CFC responsive to human-specific cytokines were assayed from the light-density livers cells harvested from 5 transplanted and 1 untransplanted NOD-SCID mice. Mice were analyzed 30 days after transplant with 1×107 hFBM cells of 23 weeks' gestation. Lines indicate the total number of CD34+/++ and CD133+CD34++ cells (right axis) shown on top of a bar chart of colony numbers (left axis). (D) A photomicrograph of representative myeloid colonies grown from liver cells shows both a large HPP-CFC-derived colony and smaller LPP-CFC-derived colonies. The size of the colonies can be gauged from the 2 mm grid shown in the background.
Figure 5.
Liver engraftment by different sources of HSCs.
(A) Engraftment of liver CD34+ cells after transplantation with 1×105–2×106 hFL or 2×107 hFBM cells. Also shown is hepatic CD34+ cell reconstitution by secondary (2°) transplanted hFL cells obtained from the BM of the primary recipients. (B) Liver CD34+ cell engraftment is compared in NOD-SCID and NSG mice. Mice were analyzed 30 days after transplant with 1×107 hFBM cells of 18 or 20 weeks' gestation. Data represent the frequency of CD34+ cells among all live cells. (C) An example of a low, but detectable, level of liver hematopoiesis observed 122 days after transplantation of 2×105 UCB cells.
Figure 6.
Hematopoietic reconstitution following transplantation of chimeric liver cells.
(A) Phenotypic analysis of light density liver cells pooled from 9 mice harvested 89 days after transplant with hFBM cells reveals evidence of CD38−CD34++ and CD133+CD34++ cells. These cells were used for transplantation into secondary recipients. (B) An example of the multilineage reconstitution of the BM of a secondary recipient 69 days after transplantation with chimeric liver cells. Arrows identify CD38−CD34++ and CD133+CD34++ candidate HSCs and CD7++CD56+ NK cells.
Figure 7.
Hematopoietic stem and progenitor cells are present in the livers of old mice.
Phenotypic analyses of hematopoietic stem cells and progenitors present in the light-density fraction of BM and livers of 2-year old Balb/cJ or 1-year old C3H/HeJ mice (A). Arrows identify populations of either CD48−CD150+ or Sca-1+CD117+ cells. Data depicted include live, single cells based on lack of PI staining and low lineage expression. Data from Balb/cJ and C3H/HeJ strains are compared to a negative isotype control shown in the left column. The Sca-1+CD117+ population contains a CD48−CD150+ population in both the BM and liver of old mice (B). Events were gated for Sca-1+CD117+Lin− single-live cells. Myeloid CFU-c were measured among light-density cells harvested from livers of 1-year old Balb/c and C57BL/6 mice (C). CFU-c per liver was calculated based on the frequencies of CFU-c shown and cell counts. Results are shown as the mean measurements on 3 or 4 mice of each strain. Note that the graph on the right represents data shown on a logarithmic scale.
Figure 8.
Hematopoietic reconstitution of uPA-NOG mice.
(A) Adult mice were transplanted with erythrocyte-depleted hFL cells by intra-splenic injection. No irradiation was used for pre-transplant cytoablation. Engraftment was evaluated 75 - 82 days after transplant. Digested liver cell suspensions were separated into quickly-settling high-density and the remaining, low-density, cells. The light-density cells were analyzed for hematopoietic reconstitution. Note the presence of CD19+ B-cells, CD33+ myeloid cells, possible immature erythroid elements (arrow, CD71++CD235a+ cells) and CD34+ hematopoietic stem (CD38−CD133+) and progenitor cells (CD38+CD133−). (B) Multilineage hematopoietic engraftment was also observed in the BM of a uPA-NOG mouse. (C) High-density cells isolated from uPA-NOG transplanted mice contained CD45−CD14+ cells likely representing liver endothelial cells as well as CD45+ hematopoietic cells (C). The same population of CD45−CD14+ cells was much less prevalent in NSG mice.
Figure 9.
Human cells engrafted in the mouse liver.
Human B2M+ cells (green) are seen growing as colonies in the parenchyma (top row). These cells are small elongated cells located around mouse hepatocytes and lining sinusoids, indicative of LSECs. Human CD45+ leukocytes (green) are found dispersed throughout the liver parenchyma as well as in some blood vessels (middle row). Note the presence of small clusters of leukocytes. LSECs stain brightly for CD34 (green) and are shown to be in close contact with the human CD45+ leukocytes (bottom row). Mouse cells are stained in the top two rows using anti-H-2Kd (red) and blue staining represents nuclei stained with DAPI. The fold-magnification used is indicated for each photograph.