Human AML Stem Cells Remain Quiescent and Exhibit Chemotherapy Resistance within the Bone Marrow Endosteal Region.

AML is characterized by the clonal expansion of immature myeloblasts initiating from rare leukemic stem cells (LSCs). We developed a primary human AML xenotransplantation model using newborn NOD/SCID/IL2rg−/ − mice. From 9 patients, 4x10⁶ T cell-depleted BMMNCs were injected iv into sublethally irradiated recipients. At 3 months, newborn NOD/SCID/IL2rg−/ − recipients demonstrated superior engraftment efficiency compared with adult NOD/SCID/IL2rg−/ − and newborn NOD/SCID/b2m−/ − recipients (37.8%, 11.9%, 12.9%, respectively; p<0.005). When sorted primary AML hCD34+CD38− cells from 6 patients were injected iv, as few as 10³ cells engrafted dose-dependently. Transplanted hCD34+hCD38− cells gave rise to hCD34+hCD38−, hCD34+hCD38+ and hCD34− cells. In serial transplantation, injection of 10³ purified hCD34+hCD38− cells resulted in long-term engraftment, giving rise to hCD34+hCD38− LSCs and hCD34+hCD38+/hCD34− blasts, demonstrating self-renewal and differentiation capacities of primary LSCs in vivo. We next examined homing and localization of LSCs using this model. In recipient femoral sections, 94.0% and 78.4% of hCD34+ cells were found in the endosteal region at day 3 and at 4 months after iv injection, respectively (10,065 cells and 24,973 cells examined, respectively; p<0.001 for each), demonstrating that human primary LSCs preferentially home to and engraft in the BM endosteal region. Dual FISH analysis using mouse and human pan-centromeric probes confirmed the human origin of these cells. When AML-engrafted mice were treated with cytosine arabinoside (Ara-C) to examine the effect of anti-leukemia drugs in vivo, PB AML burden became markedly reduced by day 8, followed by AML relapse by days 16–28. Apoptosis analysis of day 3-post-Ara-C BM demonstrated that hCD34+hCD38− LSCs are more resistant to Ara-C induced apoptosis compared to hCD34+hCD38+ and hCD34− cells (Annexin V-7AAD-: 77.1%, 33.3%, 27.5% respectively; n=3 for each; p<0.05 for each). TUNEL staining using day 3 post-Ara-C femoral sections demonstrated that TUNEL-negative cells were preferentially located at the endosteal surface while the cells in the central BM cavity were apoptotic. The cells abutting the endosteum were hCD34+hCD38− and were adjacent to osteopontin+ osteoblasts. % of cells in the G0 phase of cell cycle was significantly greater in the hCD34+hCD38− compared with the hCD34+hCD38+ and hCD34− fractions (n=5 for each; p<0.005 for each), suggesting a potential mechanism underlying relative chemo-resiatance of LSCs. In summary, primary human LSCs preferentially engraft in the BM endosteal areas where they are protected from AraC-induced apoptosis. Cell cycle quiescence of LSCs may be one of the mechanisms underlying chemoresistance of LSCs.