Since T-cell deficient “nude” (Foxn1
mutation) mice were identified in 1970, xenogeneic grafting of normal and pathologic human cells and tissues to immune deficient mice has been embraced as an approach to move in vitro
models to more complex in vivo
. Success in humanizing mice with normal tissues moved significantly forward when the severe combined immunodeficient T-B-mice (Scid, Prkdc
mutation) were identified and found to support human hematopoietic cells and lymphoid organs 73,74
. Sequential improvements have given several relatively simple and reproducible mouse models for generation of a ‘human immune system’ (HIS) mice. Strains now commonly used to prepare HIS mice are NOD-SCID-Il2rg−/−
(NOG) and BALB/c-Rag2−/−Il2rg−/− 75,76
and protocol refinements continue 75,77
HIS mice have been useful for studying pathogens such as HIV that directly target the human lymphohematopoietic cells, including cells found in the female reproductive tract 78
. However, human lymphocyte lineage differentiation in HIS mice differs markedly. B cell reconstitution is robust, T cell reconstitution is reasonable, but NK cell and myeloid lineage reconstitutions are generally poor to undetectable 79
. Since IL-15 trans-presentation regulates endogenous human NK cell homeostasis, use of IL-15 receptor agonists has been recommended to improve xenogeneic NK cell engraftment 77
. No current model is suitable for addressing the question of whether xenogeneically engrafted human NK cells home to the gestational uterus and effect spiral arterial modification. This is because the models employ recipient preconditioning by irradiation (between 320 and 375 cGy) rendering the recipient reproductively sterile. We turned to 5-fluorouracil (FU), a thymidylate synthase inhibitor as a preconditioning agent for of 6 wk old female BALB/c-Rag2−/−Il2rg−/−
mice, at a dose of 150mg/kg 80
. After 24 hr, CD34+
cord blood cells enriched by negative selection were inoculated. Six weeks later the females were bred and euthanized for study.
The choice of xenograft recipient is important. Because our research question is focused on promotion of decidual angiogenesis and spiral arterial modification, we are not able to use recipients with a NOD background because the decidual arterial in NOD mice is abnormal 81
. Our syngeneic mouse to mouse grafting of Rag2−/−Il2rg−/−
mice on either the C57BL/6 or BALB/c is consistently successful in establishing fully functional, graft-derived uNK cells 82,83
that effect quantifiable spiral arterial modification, we selected preconditioned BALB/c-Rag2−/−Il2rg−/−
for study and used 16 as recipients for 1×105
cord blood cells. Variables compared were:
- Administration of freshly isolated cells versus cells expanded in culture (24 hr in StemSpan SFEM medium with CC100 Cytokine Cocktail) 84,85.
- Tail vein (IV) versus intrafemoral (IF) cell injection 86,87.
- Without or with human IL15/IL15Rα complex treatment at 6 and 7 wks of age and at gd6.5 after mating 77. These four females were paired with males immediately after their 2nd injection and bred within a few days.
Two additional variables are present in these studies. The first is placenta donor variability—three placentae were used. The second is whether the prepared females successfully mated and subsequently conceived.
All mice were killed at gd12.5 with sera and organs collection for quantification of human IgG by ELISA, RT-PCR for human chromosome 17-specific α-satellite DNA and histology. summarizes our progress to date. Of the 16 females used in this study, 14 became pregnant and carried a viable litter to gd12.5. There were no differences in implantation site numbers between unmanipulated BALB/c-Rag2−/−Il2rg−/− and hu-CD34+ cell inoculated BALB/c-Rag2−/−Il2rg−/− (). Of the 14 pregnant females, 3 were identified as chimeric in their implantation sites (decidual and MLAp) and peripheral organs (maternal liver and spleen) and their sera contained huIgG (, ). One of the two mice who mated but were not pregnant was also chimeric in spleen and liver and had circulating hu-IgG. Cells prepared from one of the 3 donor placentae gave no reconstitution.
Summary of BALB/c-Rag2−/−Il2rg−/− mice engrafted with human cord blood CD34+ cells at gestation day 12
Fig 4 Implantation sites outcomes after human CD34+ cell inoculation. A total of 16 BALB/c-Rag2−/−Il2rg−/− mice were pretreated with 5-FU (150mg/kg) and inoculated with 1×105 with human CD34+ cord blood cells. Six wks (more ...)
Implantation site histology in the pregnant, chimeric mice was quite variable between littermates, an observation we have never made in unmanipulated or syngeneically-transplanted females. It also differed to what was anticipated (). Cells with a lymphoid appearance were not present in the decidua basalis and there was a gain in spiral arterial pathology. In the most severely altered sites (), a greatly enlarged vascular wall surrounded the spiral arteries. This region showed a localized loss of reactivity with many histochemical stains and appeared to have lots all of its collagen fibers which are eosin-reactive in normal BALB/c and BALB/c-Rag2−/−Il2rg−/− mice (). This region is not an artifact and has been seen multiple times in another series of intrahepaticly inoculated neonatal recipients (M. Bilinski and B. A. Croy, data not shown). A littermate implantation site to the one illustrated had much milder changes. In it, individual large, irregularly shaped, pale staining mononuclear cells with low nuclear to cytoplasmic ratio were seen that appeared to have specifically homed to the spiral arterial walls. The unusual cells could not be stained by PAS, Alcian blue or Masson’s Trichrome indicating lack of glycoprotein, collagen and mucopolysaccharides (not shown). We anticipate but as yet have no evidence that the unusual cells and tissue are of human origin because 5-FU treatment followed by syngeneic grafting does not induce this appearance. Neither does administration of 3 doses of the IL15/IL15Rα complex alone without cells (not shown). The unusual cells we report are not reactive with antibodies to human CD45 and may represent progeny of circulating human mesenchymal or other stem cells not removed by the CD34− cord blood selection procedure. It is of interest that these proven chimeric implantation sites do not reveal an immune graft versus placenta pathology and that if the fetus imaged in () was doomed to die in late gestation, its compromise would have been effected through the maternal vasculature with particular involvement at the spiral arterials. That components of human decidual tissue beyond immune cells and circulating endothelial progenitor cells might arise from circulating progenitor cells is a novel hypothesis suggested by these preliminary studies. Might it also be possible that the unusual, spiral artery-homed cells we detected are the early progenitors of uNK cells and that uNK cells arise from committed lymphoid cells, and in humans from circulating CD56bright cells is in error? Readers will realize that we have not yet shown that the unusual cells depicted in are human in origin and that we remain far from our goal of a humanized pregnant mouse model that will enable in vivo studies of the interactions between human uNK cells and the stromal cells comprising the spiral arteries.
Fig 5 Photomicrographs comparing gd12 implantation sites from wildtype BALB/c (A, a), BALB/c-Rag2−/−Il2rg−/− (alymphoid; B, b) and human CD34+ cell-engrafted BALB/c-Rag2−/−Il2rg−/− mice (iv injection, (more ...)