Improved Engraftment of Human Hematopoietic Stem Cells in NOD/SCID Mice by the Antioxidant, N-Acetyl-L-Cysteine
Authors: Hu Linping , Gao Yingdai , Liu Yanfeng , Cheng Hui , Xu Jing , Yang Ming , Yuan Weiping , Cheng Tao 
Published: October 2019

Abstract
Abstract 1884
Non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice are widely used as a human-mouse xeno-transplant model for assessing engraftment of human hematopoietic stem cells (HSCs). Optimizing this model and understanding the variables that affect engraftment are critical to correctly interpret the engraftment results of human HSCs in NOD/SCID recipients. However, the engraftment efficiency of human HSCs remains very low in NOD/SCID mice. Larger efforts have been devoted to improving engraftment by increasing the immune-deficiency of the mice, but less effort has been made toward other negative parameters in the host. Our preliminary study showed that NOD/SCID mice had higher levels of reactive oxygen species (ROS) in the bone marrow (BM) in comparison with other commonly used mouse strains (C57BL/6 and BALB/C). Given the previous studies by us and others showing that excessive ROS could impair the function of HSCs and that antioxidants were able to overcome the exhaustion of mouse HSCs in transplant recipients, we hypothesized that the poor engraftment of human hematopoietic cells in NOD/SCID recipients may be partially attributed to higher levels of ROS in NOD/SCID BM and a reduction of ROS by antioxidants may improve the engraftment of human HSCs in NOD/SCID mice.
To test this hypothesis, NOD/SCID (8- to 12-week-old) mice were injected subcutaneously daily with an antioxidant, N-acetyl-L-cysteine (NAC) or PBS (control) for two weeks before being irradiated with 200 cGy from a cesium-137 source at 70cGy/min. Different doses of CD34+ cells or highly-enriched HSCs from human cord blood (CB) were injected through the tail vein or into the right tibia of the mice. The mice were maintained on NAC treated drinking water following injection, then sacrificed 12–14 weeks after transplantation to measure the engraftment levels of different hematopoietic cell lineages.
We found that treatment with NAC was able to lower the levels of ROS in NOD/SCID BM. At the highest dose of injected CD34+ cells (>5×105), the NAC treated recipients displayed a significant increase of engraftment (2.1-fold) when compared with the control group (Control vs. NAC treated recipients: 11.04±3.11% vs. 23.21±4.0%, p=0.0224; n=20/each). This improvement was even more significant when injected cell numbers were reduced (2×105 and 1×105 had 3.9- and 4.9- fold higher engraftment, respectively, in NAC treated recipients), thus suggesting that saturating levels of HSCs may ease the anti-oxidant effect on engraftment. Furthermore, we also demonstrated higher levels of overall engraftment and multi-lineage differentiation of human HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+Rholow) with a limiting dilution analysis. In comparison with the control mice, NAC treated recipients displayed 2.5-, 3.5-, and 5.7-fold increases in engraftment in the injected tibia (IT), BM and spleen, respectively. The frequency of SCID-repopulating cell (SRC) in IT was approximately 3.0-fold higher in NAC treated mice than in control mice (1 in 108 vs. 1 in 36). Similar improvements (4.1- to 7.9- fold) in SRC frequencies were also detected in BM and spleen. Notably, NAC increased the probability of positive engraftment when a single human HSC was directly transplanted into the BM of NOD/SCID mice. In summary, our current study uncovers a previously unappreciated negative effect of ROS in the human-NOD/SCID xenotransplant model and reduction of ROS via antioxidants such as NAC may significantly enhance the engraftment of human hematopoietic stem cells in NOD/SCID mice.

Disclosures:
No relevant conflicts of interest to declare.