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  • Introduction The NOD SCID Il

    2018-11-14

    Introduction The NOD.SCID Il2rγ (NSG) mouse strain dramatically improved investigators’ ability to study human hematopoietic stem cell (HSC) engraftment (Rongvaux et al., 2013; Shultz et al., 2005). The NSG strain combines a series of mutations that inhibit the host’s immune system by different mechanisms (Bosma et al., 1983; Cao et al., 1995; Greiner et al., 1995; Ohbo et al., 1996; Shultz et al., 1995). The NSG and other host strains require myeloablative conditioning (i.e., radiation) to achieve high levels of human chimerism (McDermott et al., 2010; Shultz et al., 2005). In addition to the effects of irradiation on the hematopoietic system that can promote engraftment (Broudy, 1997), confounding side effects of irradiation, including necrosis and apoptosis of gastrointestinal, neural, and muscle tissues, can lead to wasting, infection, and even death (Li et al., 2004; Qiu et al., 2010). Two recent studies report limited success in engrafting human HSCs into nonmyeloablated NSG hosts, but the chimerism achieved in the peripheral blood was modest (an average of 3% ± 3% and 18.3% ± 13%) (Brehm et al., 2012; Bueno et al., 2010). Viable mutant Kit oncogene (Kit) murine strains support the engraftment of mouse hematopoietic dna staining without host irradiation (Wang and Bunting, 2008). The Kit (i.e., c-Kit, stem cell factor [SCF] receptor) encodes a type I membrane protein in the type III tyrosine kinase growth factor receptor family (Yarden and Ullrich, 1988), which is expressed on hematopoietic, melanocyte, neural, and germ cells (Mintz and Russell, 1957; Poole and Silvers, 1979; Russell, 1979). When its ligand, SCF, binds to c-Kit, it induces receptor homodimerization and signal transduction (Hsu et al., 1997). The c-Kit is required for normal hematopoiesis, and viable mutants most closely resemble aplastic anemia (Geissler et al., 1981). Mouse hosts with mutations in Kit thus provide a competitive advantage for WT donor cells and allow the engraftment of HSCs with reduced or no irradiation (Fleishman, 1996; Waskow et al., 2009). Until recently, these strains have been short-lifespan heterozygotes (e.g., Kit), causing a burden on producing experimental hosts (Cosgun et al., 2014; Waskow et al., 2009). We demonstrate that nonirradiated NBSGW mice are similar in chimerism to that achieved in irradiated NSG (irNSG) mice and that NBSGW mice also support the serial transplantation of HSCs.
    Results
    Discussion In this study, we generated mice homozygous for Prkdc, Il2rγ, Kit, and NOD Sirpa alleles and showed that the resultant NBSGW model supports robust levels of humanization in peripheral blood, bone marrow, and spleen in the absence of irradiation. The observed levels of de novo derived human HSCs in NBSGW mice are significantly higher than the values previously reported for unconditioned NSG or NSG-transgenic mice (Brehm et al., 2012; Bueno et al., 2010). In our studies, the unconditioned NBSGW strain supported levels of human chimerism in peripheral blood that was 9-fold higher (average 61% versus 8.3%) compared with the unconditioned NSG strain. Furthermore, these levels are equivalent to those recently reported in multiple irradiated Rag2Il2γ mice possessing humanized loci for TPO, CSF1, IL3, and CSF2 in which hematopoietic development is enhanced (Rongvaux et al., 2011, 2014). We further demonstrate, through serial transplantation, robust normal development of human HSCs in NBSGW marrow and the developing HSCs ability to repopulate a secondary recipient. To our knowledge, Kit mutants when combined with NSG mice yield the highest level of human hematopoietic chimerism obtained in any mouse strain without conditioning by ionizing radiation. While this manuscript was in review, four Kit mutant strains on either the BALB/c RAG1 (BRg Kit, BRgWv) or NSG (NSG Kit, NSGWv; NSGWv/+, and NSGW41) backgrounds were described (Cosgun et al., 2014). These strains are similar to the NBSGW model exhibiting comparable humanization potential in the absence of irradiation. Both BALB/c and NOD immunodeficient-based strains support varied types of humanization. However, NOD background strains have higher humanization potential (Brehm et al., 2010) primarily resulting from polymorphisms in the Sirpa gene that prevents the NOD macrophages from engulfing human cells (Takenaka et al., 2007). Hence, the Kit mutant strains NBSGW, NSGWv, NSGWv/+, and NSGW41 are better suited recipients when compared with the BRgWv strain. Homozygous Kit NSG mice have a low life expectancy, making the NSGWv/+ the more useful of the two for long-term experiments (Cosgun et al., 2014). However, genotyping to identify suitable NSGWv/+ recipients among litters is time consuming.