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    • Our system is very similar to

    2018-11-07

    Our system is very similar to that developed by the Poss Lab using the same ubiquitin promoter fragment (Chen et al., 2013), although their study did not use sedation routinely for BLI acquisition and allowed animals to swim freely (donor BLI was assessed 28 days post-HCT). By optimizing sedation and keeping the animals immobile, we were able to measure the fraction of hematopoietic estrone that homed to the WKM 24 hr post-HCT, a unique feature of our approach. BLI-based studies in mice using luciferase-expressing donor cells can also measure homed cells as early as 24 hr post-HCT, but the spatial-temporal location of the migrated cells is not as precise as the studies we have performed (Wang et al., 2003). In our first screen, we identified a compound, ergosterol, as a positive mediator of hematopoietic cell homing in zebrafish and mice. Ergosterol is a sterol family member produced by mushrooms and fungi in significant quantities. After UVB irradiation, ergosterol is converted to vitamin D2 (ergocalciferol), which is a vitamin D analog. Unlike vitamin D3, D2 is not readily converted into 25 hydroxyvitamin D, the precursor to the active form of vitamin D, 1,25(OH)2D3. While historical data suggest that vitamin D2 does bind to the VDR with a similar affinity as vitamin D3 metabolites, vitamin D2 has less vitamin D binding protein affinity, shortening its plasma half-life, and it has been suggested that it is not equivalent to vitamin D3 in potency (Hollis, 1984; Houghton and Vieth, 2006). Our data indicating that ergosterol does not act through VDR also suggest an alternative to the classic vitamin D3-VDR pathway. Prior to this report, ergosterol had not been studied in the context of hematopoiesis. Although other vitamin D family members have been shown to have cell growth-promoting effects on the hematopoietic system and have been specifically utilized in the management of anemia (Christakos et al., 2013; Erturk et al., 2002; Hall and Juckett, 2013; Hewison et al., 2001). In addition, Cortes et al. (2016) have recently shown that vitamin D3 has HSPC growth-promoting effects in embryonic zebrafish as well as in human umbilical cord blood. Furthermore, ergosterol may also have cytoprotective effects, as rats that receive ergosterol prior to carbon-tetrachloride-induced liver injury have a significant reduction in liver injury, inflammation, and fibrosis (Peng et al., 2014). These data are consistent with our finding that cells exposed to ergosterol have improved viability. There are increasing data that vitamin D plays a positive role in HCT (Hall and Juckett, 2013). Hansson et al. (2014) recently measured vitamin D levels (total vitamin D including D2 and D3) in children prior to HCT. They found that children who were vitamin D-sufficient pre-HCT had more robust neutrophil engraftment at day 30 post-HCT (the first time point measured) and lower rates of hematopoietic cell rejection. Vitamin D-sufficient children with malignancy also had improved overall survival (87% versus 50%, p = 0.01). Other groups have shown that severe vitamin D deficiency at HCT was associated with reduced survival after transplant in pediatric patients (Wallace et al., 2015). These reports suggest that vitamin D positively affects engraftment and overall HCT outcome. The mechanisms for the enhancement of engraftment were not demonstrated, but our data suggest that perhaps vitamin D family members can increase CXCR4 expression, one of the key mediators of homing after transplant. In this study, we demonstrated BLI to be a useful tool to non-invasively track transplanted hematopoietic cells in the zebrafish, allowing the homing/engraftment process to be observed in a living adult recipient. The quantified luminescent signals were consistent and corresponded well with the number of cells engrafting. Our longitudinal analysis demonstrated the kinetics of donor WKM cells showing a high level of engraftment by short-term progenitor cells, and lower, stable level of engraftment by more rare long-term repopulating stem cells. Using BLI, we were able to perform a chemical screen of compounds to find those that enhance cellular homing. We found that ergosterol, a phytosterol and vitamin D2 precursor, improves homing of hematopoietic cells in zebrafish and mouse models. The mechanism of action is incompletely understood, although CXCR4 upregulation plays a role and likely involves pleiotropic effects on cell expansion and survival.