Acute myeloid leukemia (AML) was the first disease in which leukemia-initiating cells (LICs) were identified using the NOD/SCID (NS) xenotransplantation model. 1, 2 These cells were originally reported to be rare. 2 Recent data in melanoma indicate that the frequency of tumor-initiating cells (TICs) increases dramatically when more permissive immunodeficient NS/interleukin-2 receptor γ-chain null (NSG) mice are used, 3 and contrary to the findings of Boiko et al., 4 TICs in melanoma might not be purified, 5 thus questioning the existence of TICs in melanoma. A study of pancreatic, non-small cell lung, and head and neck carcinoma shows that despite a 10-fold increase in the frequency of TICs in NSG compared with NS, the frequency of TICs remains low. 6 Recently, we demonstrated that LICs are also present in the CD34þCD38þ fraction, 7 contrary to our initial findings. 2 We therefore re-investigated the issue of the frequency of LICs, comparing the originally used NS and the new NSG model.

Here, we first report that 14 AML samples, which did not engraft in NS mice, could still not engraft in NSG (see Supplementary Table 1a and Supplementary Figure 1). Despite recent data, 8–10 including our own, showing that higher level of engraftment could be achieved in NSG compared with NS mice, the NSG model does not seem to be significantly more permissive, by enabling the engraftment of ‘non-engrafter’AML samples tested originally in NS. 11 For patients with AML that engraft (see Supplementary Table 1b), we estimated the LIC frequency using limiting dilution analysis. Figure 1 shows the results of 11 AML samples in which the frequency of LICs was compared side by side between NS and NSG (Figure 1 and Supplementary Table 2). It thus appears that the frequency of LICs varies between patients in both models. For patients in whom the frequency of LICs in NSG was higher than 1 in 50 000 cells (patients 6 to 11), the absolute number of LICs increased by 12-to 111-fold in NSG, compared with NS. This difference was less than five-fold for patients with lower frequency (LICs> 1 in 50000; patients 1 to 5). Our results suggest that when lower numbers of cells were injected, a more pronounced difference was seen between the two models. Recently, we showed that the residual immune cells present in NS have a profound effect on the engraftment of antibody-labeled and-sorted CD38þ cells. When using an anti-natural killer cell treatment or a more permissive NSG model, a drastic reduction of the clearance of these CD38þ cells7 was seen. Consequently, we decided to