Low-Dose Busulfan Reduces Human CD34+ Cell Doses Required for Engraftment in c-kit Mutant Immunodeficient Mice

Abstract

Humanized animal models are central to efforts aimed at improving hematopoietic stem cell (HSC) transplantation with or without genetic modification. Human cell engraftment is feasible in immunodeficient mice; however, high HSC doses and conditioning limit broad use of xenograft models. We assessed human CD45+ chimerism after transplanting varying doses of human CD34+ HSCs (2 × 105 to 2 × 106 cells/mouse) with or without busulfan (BU) pretransplant conditioning in c-kit mutant mice that do not require conditioning (non-obese diabetic [NOD]/B6/severe combined immunodeficiency [SCID]/ interleukin-2 receptor gamma chain null (IL-2rγ−/−) KitW41/W41 [NBSGW]). We then tested a range of BU (5–37.5 mg/kg) using 2 × 105 human CD34+ cells. Glycophorin-A erythrocyte chimerism was assessed after murine macrophage depletion using clodronate liposomes. We demonstrated successful long-term engraftment of human CD34+ cells at all cell doses in this model, and equivalent engraftment using 10-fold less CD34+ cells with the addition of BU conditioning. Low-dose BU (10 mg/kg) was sufficient to allow human engraftment using 2 × 105 CD34+ cells, whereas higher doses (≥37.5 mg/kg) were toxic. NBSGW mice support human erythropoiesis in the bone marrow; however, murine macrophage depletion provided only minimal and transient increases in peripheral blood human erythrocytes. Our xenograft model is therefore useful in HSC gene therapy and genome-editing studies, especially for modeling in disorders, such as sickle cell disease, where access to HSCs is limited.

An Overview of Different Strategies to Recreate the Physiological Environment in Experimental Erythropoiesis

“…Nevertheless, important discoveries on bone marrow and microenvironments have been made in recent years, providing a better understanding of the physiological environment of erythropoiesis that may allow us to solve this problem. As a result, promising new tools have been developed, ranging from simple erythroid culture to 2D or 3D co-culture, allowing an increase in: the long-term expansion time of erythroid cells, the production of red blood cells, enucleated cells, and the presence of adult hemoglobin [18,20,32,42,68,113,114].…”

“…Based on previous results [39,40], they designed a loss-of-function of KIT receptors into NOD/SCID Il2rg −/− mice, which carry out ineffective erythropoiesis. This new tool provides higher and stable engraftment but also enhanced HSPCs differentiation and enucleation compared to NSG basic mice [38,41,42]. To avoid the phagocytosis of hRBCs by murine macrophages, they used clodronate-containing liposomes (CloLip) to eliminate them from the blood.…”

Preclinical evaluation for engraftment of CD34+ cells gene-edited at the sickle cell disease locus in xenograft mouse and non-human primate models

“…The next day, gene-edited SCD CD34+ cells were injected into 7-week-old male mice. 42 , 58 , 59 These mice received sublethal busulfan conditioning (25mg/kg, Busulfex, PDL BioPharma, Redwood City, CA, USA) 2 days before transplantation.…”

“…The next day, gene-edited SCD CD34+ cells were injected into 7-week-old male NOD/B6/SCID/IL2rγ −/− /Kit W41/W41 mice (NOD.Cg-Kit W-41J Tyr + Prkdc scid Il2rg tm1Wjl /ThomJ, Jackson Laboratory). 42 , 58 , 59 These mice received sublethal busulfan conditioning (25mg/kg, Busulfex, PDL BioPharma) 2 days before transplantation. The use of mice in transplantation experiment (H-0228) was approved by the Animal Care and Use Committee of the NHLBI.…”

Perspectives on Non-BLT Humanized Mouse Models for Studying HIV Pathogenesis and Therapy

“…Therefore, our research group has conventionally constructed humanized NOJ mice without irradiation before HSC transplantation [ 47 , 57 , 77 , 111 ], recognizing that there is a decrease in chimerism, as indicated elsewhere [ 38 , 87 ]. To avoid such limitations, busulfan treatment is a useful alternative to irradiation for myeloablation [ 29 , 153 , 154 , 155 , 156 , 157 ], but it should be noted that higher doses of busulfan are toxic and fatal [ 153 , 156 ]. Another option is to use a non-SCID strain of mouse such as the NRG or DKO mouse, which is less sensitive to irradiation [ 2 ].…”