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    • Our goal was to generate a viable homozygous

    2018-11-08

    Our goal was to generate a viable homozygous strain that would engraft with high efficiency without irradiation. Based on previously reported Kit mutant phenotypes (Geissler et al., 1981), we hypothesized that the Kit could be maintained as a homozygous line when crossed with the NSG, and the contribution of the NSG strain would boost the efficiency of humanization in the absence of irradiation. While the NBSGW is a mixed NOD and C57BL/6J background, the Cosgun strain is a congenic NOD also incorporating a homozygous Kit allele. A direct comparison of engraftment results between the two strains is currently difficult due to differences in experimental approaches (e.g., cell numbers and sources) and reported postengraftment end points (e.g., 12 weeks versus up to 40 weeks). However, the highest levels of engraftment in the absence of irradiation were observed in homozygous Kit immunocompromised mice in both studies, potentially indicating that a mixed background is inconsequential given the correct genetic elements. Based on these arguments, either the NBSGW or the NSGW41 strains are of choice over their contemporary strains in the absence of irradiation and may prove as a widely enabling resource. In addition to the increased human chimerism in the blood, we observed a significantly greater percentage of de novo human erythroid cells in the marrow of NBSGW mice (average 26.0% versus 0.1%). These levels are ∼5–12 times higher on average than previous reports with irNSG mice (Hayakawa et al., 2010; McDermott et al., 2010). However, our analysis of peripheral blood for GlyA+ cells did not indicate we had achieved circulating human erythrocytes, suggesting that the current mouse model still lacks signals to complete human erythrogenesis. The NBSGW mice exhibit a high degree of human chimerism, albeit a bias in differentiation of the engrafted human hematopoietic cells remains. In normal human peripheral blood, granulocytes, T cells, and csf-1r are present at 53%, 20%, and 33%, respectively (Rongvaux et al., 2013), but within our study, they are present at 2.5%, 3.3%, and 68%, respectively. These biases are comparable to those previously observed in other mouse xenograft models (Brehm et al., 2010; Cosgun et al., 2014; McDermott et al., 2010) and suggest that deficiencies exist in species cross-reactivity of signaling molecules supporting typical human hematopoietic cell distributions (Rongvaux et al., 2013). Additional genetic modifications to NBSGW mice are possible with the aim of eliminating these biases. Such modifications might include replacing growth factors or matrix molecules that poorly cross-react between mouse and human species. A number of recently described strains, when crossed with NBSGW, might further improve the differentiation of human HSCs (Billerbeck et al., 2011; Brehm et al., 2012; Rongvaux et al., 2014). Although the main focus of our study was to grade the relative efficiency of human HSC engraftment in a nonirradiated mouse model, the lack of T cell production in NBSGW, NSGW41 and NSG warrants further discussion. All three strains support the production of a limited number of T cells (Cosgun et al., 2014). This may be partially due to a relatively thin thymic membrane and to the documented poor thymic microenvironment (McDermott et al., 2010; Shultz et al., 2005) for all strains. Furthermore, in this study, we measured engraftment at 12 weeks, while T cell development is building and not yet plateaued (Hiramatsu et al., 2003). However, the data and reports indicate that T cell production is elevated in the homozygous Kit strains in comparison to others (Cosgun et al., 2014). In sum, these facts create an opportunity to improve humanized murine models via the improvement of thymic function. Pluripotent stem cell (PSC) technology offers one potential route to re-establish human thymic function in humanized mouse models (Takahashi et al., 2007; Yu et al., 2007). For example, two recent publications report the correction of a developmental T cell deficiency in immunocompromised mice (Parent et al., 2013; Sun et al., 2013). After differentiating human embryonic stem cells to a thymic epithelial progenitor cell type and grafting these cells into mice, the authors produced human thymic structures capable of further development and education of both mouse and human T cells in vivo (Parent et al., 2013; Sun et al., 2013). Thus, further refinements of PSC technology may afford even better opportunities to study allogeneic and autologous human HSC transplantation in the in vivo mouse environment.