The Adult Livers of Immunodeficient Mice Support Human Hematopoiesis: Evidence for a Hepatic Mast Cell Population that Develops Early in Human Ontogeny

<div><p>The liver plays a vital role in hematopoiesis during mammalian prenatal development but its hematopoietic output declines during the perinatal period. Nonetheless, hepatic hematopoiesis is believed to persist into adulthood. We sought to model human adult-liver hematopoiesis by transplantation of fetal and neonatal hematopoietic stem cells (HSCs) into adult immunodeficient mice. Livers were found to be engrafted with human cells consisting primarily of monocytes and B-cells with lesser contributions by erythrocytes, T-cells, NK-cells and mast-cells. A resident population of CD117⁺⁺CD203c⁺ mast cells was also documented in human midgestation liver, indicating that these cells comprise part of the liver's resident immune cell repertoire throughout human ontogeny. The murine liver was shown to support human multilineage hematopoiesis up to 321 days after transplant. Evidence of murine hepatic hematopoiesis was also found in common mouse strains as old as 2 years. Human HSC engraftment of the murine liver was demonstrated by detection of high proliferative-potential colony-forming cells in clonal cultures, observation of CD38⁻CD34⁺⁺ and CD133⁺CD34⁺⁺ cells by flow cytometry, and hematopoietic reconstitution of secondary transplant recipients of chimeric liver cells. Additionally, chimeric mice with both hematopoietic and endothelial reconstitution were generated by intrasplenic injection of immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene. In conclusion, the murine liver is shown to be a hematopoietic organ throughout adult life that can also support human hematopoiesis in severely immunodeficient strains. Further humanization of the murine liver can be achieved in mice harboring an uPA transgene, which support engraftment of non-hematopoietic cells types. Thus, offering a model system to study the interaction of diverse human liver cell types that regulate hematopoiesis and immune function in the liver.</p></div

Hematopoietic stem and progenitor cells are present in the livers of old mice.

<p>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.</p

Hematopoietic reconstitution following transplantation of chimeric liver cells.

<p>(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.</p

Human hematopoiesis in the livers of mice transplanted with fetal cells.

<p>(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.</p

Hematopoietic reconstitution of uPA-NOG mice.

<p>(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.</p

Mature human blood cells in the murine liver.

<p>(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.</p

Human cells engrafted in the mouse liver.

<p>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-2K^d (red) and blue staining represents nuclei stained with DAPI. The fold-magnification used is indicated for each photograph.</p

Liver engraftment by different sources of HSCs.

<p>(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.</p