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point (Figure 6D), whereas the presence of MSC did not appear to improve engraftment regardless of the origin of the MSC (Figure 6D).
Discussion
Ongoing development of so-called “humanized” mouse strains that combine immunodeficiency with expression of human proteins or cells permits increasingly sophisticated modeling of human diseases in xenotransplantation mod- els. Here we have used the NSG and NSG-S strains to study the effects of human cytokines on engraftment and growth of human myeloid diseases, AML and MDS. Our results show that NSG-S mice represent a significantly improved patient-derived xenotransplantation model to accelerate and enhance leukemic engraftment compared to NSG mice, and to support engraftment for the vast majority of all pri- mary AML samples, making this model particularly useful for pre-clinical studies. In contrast, engraftment of human MDS in NSG or NSG-S mice is possible but not robust and in several cases not sustained. High-risk MDS is more likely to achieve long-term engraftment compared to low-risk MDS. The expression of human cytokines in NSG-S mice marginally improves engraftment levels. In developing this model we also note that human MSC do not provide long- term MSC engraftment in NSG-S mice and do not con- tribute to MDS cell engraftment or expansion. Overall, these results are consistent with in vitro studies suggesting that AML cells are actually more responsive to bone mar- row-derived cytokines than MDS cells, at least as measured by engraftment and growth in immunocompromised mouse strains.29-32
Engraftment of AML samples in NSG mice has been the standard measure of assessment of leukemic stem cells for the last ten years. However, our results with AML overall, and particularly with inv(16) AML, demonstrate that severe combined immunodeficiency leukemia engrafting ability, the functional definition of leukemic stem cells, can be strongly affected by the recipient mouse strain and the extent to which the recipient environment contains human cytokines or cytokines that cross-react with human cytokine receptors. More formal exploration of stem cells comparing NSG and NSG-S mice will further define the dependency of severe combined immunodeficiency leukemia-initiating cells on cytokines. Furthermore, these studies raise significant questions about AML biology that should be addressable using the NSG-S model. Previous authors have suggested that AML cells require pathological activation of signaling pathways for full transformation.33 However, our data demonstrate that the majority of AML samples may remain responsive to cytokines produced by the bone marrow as was also shown by Ellegast et al.34 Given the recent results that effective 3rd-generation FLT3 inhibitors are clinically active but not curable it may be of therapeutic value to consider whether cytokines can regu- late cell survival allowing AML cells to escape FLT3 inhibi- tion.35
In contrast to AML, human MDS xenotransplantation has not been thoroughly studied. Although we studied a modest number of MDS samples, our results comparing MDS injections into NSG versus NSG-S mice with or with- out MSC co-injection are similar to other recently described results.36 A critical question is the formal definition of an MDS-engrafting cell, the functional equivalent of the severe
combined immunodeficiency leukemia-initiating cell as ini- tially defined by Bonnet and Dick.22 AML stem cells typical- ly traffic to the marrow after xenotransplantation, remain quiescent for some weeks and then initiate expansion. In contrast, MDS cells in our study were injected into the mar- row where they appear to remain but do not expand for several months. Some samples, from patients with high- risk MDS, demonstrated expansion and our results show that MDS initiating cells can be maintained in NSG-S mice. More specifically, secondary recipient NSG-S mice injected with human CD45 cells isolated from primary recipient mice developed abnormal hematopoiesis identical to that of the primary recipients, accompanied by a significant expansion of the phenotypic stem/progenitor cell compart- ment, which is typical in cases of AML passaged into sec- ondary xenotransplanted animal models. Whereas genetic analysis showed the presence of identical genetic lesions between the patients’ samples and the primary recipient animals (data not shown), the selection of an unidentified minor AML clone in the secondary recipient animals is a possibility that requires further exploration. Examination of the human cell populations present in the murine bone mar- row revealed signs of abnormal hematopoiesis reflected by the presence of stem/progenitor cells, the prevalence of the myeloid component and, in the majority of cases, the com- plete absence of lymphoid cells. Erythropoiesis was com- promised and even though cells at the initial stages of human erythroid differentiation (ERY1, ERY2) were present in both the bone marrow and the spleen (data not shown), these cells were not capable of further differentiation, sug- gesting a blockage in the erythroid differentiation process, as has been previously described.37 Our findings suggest that the development of a patient-derived xenotransplanta- tion model for MDS is possible, but not yet robust, and that the definition of MDS stem cells may include long-term engraftment but not expansion. The development of an ectopic human niche might provide a solution since prom- ising results have been reported in several types of leukemia.38,39
The use of MSC to humanize the murine bone marrow niche has been proposed as a method to enhance MDS engraftment.20 However, we demonstrated that human MSC engrafted into murine bone marrow do not establish long-term engraftment. It is theoretically possible that short-term survival of human MSC may promote engraft- ment of MDS cells but this was not seen in our studies. The reason for this discrepancy from other studies is not clear, but it is notable that AML engraftment is known to be high- ly variable among animal colonies.
In summary, AML cells demonstrate enhanced engraft- ment in NSG-S mice compared to NSG mice. MDS cells do not demonstrate a similar response to either the human cytokines produced by the NSG-S strain or by co-injection of human MSC. The findings of these studies are consistent with those of previous in vitro studies of AML and MDS and may suggest that MDS cells are not capable of responding to human cytokines with an increase in survival or growth. Further work may determine whether this is a fundamental aspect of the ineffective hematopoiesis that defines MDS.
Acknowledgments
We thank Lucio Castilla (University of Massachusetts, Worcester, MA, USA) for his assistance in analyzing inv(16) and April Schrank-Hacker for performing FISH studies. We also thank Joy Cannon, Rebecca Kotcher and the clinical staff for consenting
haematologica | 2018; 103(6)