Direct Confirmation of Quiescent Property of AML Stem Cells by Showing Markedly Decreased Plating Efficiency in Single Leukemic Stem Cell Culture

Cancer stem cell, recently discovered to exist in colon cancer and brain tumor, is resistant to anti-cancer drugs and radiotherapy, demanding the development of new drugs and treatment strategies targeting tumor stem cells. Leukemic stem cell (LSC) has been accused to play a pivotal role in pathogenesis of hematological malignancy such as acute myeloblastic leukemia (AML). Various anti-cancer medicines, particularly anti-proliferative agents, have been ineffective in treating LSC due to its slower division process and longer interphase compared to normal stem cell and hematopoietic cell. This study comparatively examined growth and proliferation capacity (plating efficiency) of clonogenic hematopoietic progenitors and LSC from healthy donors and AML patients using single cell sorting and culture system (BD FACS Aria cell sorter; BD Biosciences, San Jose, CA). A total of 384 normal hematopoietic stem cells (CD34+CD38+/CD38−) were obtained from peripheral bloods and cord bloods donated by four donors using single cell sorter, and individual single cells were cultured in 96-well plates with each well containing 100ul of serum media, 100ng/ml of stem cell factor, 100ng/ml of Flt-3, 100ng/ml of thrombopoietin and 50ng/ml of G-CSF for five days. 768 single LSC (CD34+CD38−) and 384 single CD34+CD38+ cells were obtained from three AML patients. Growth and proliferation capacities of normal hematopoietic stem cell and LSC were determined in terms of plating efficiency (number of the wells in which more than two cells grew/total number of cells in 96-well plate culture × 100). Plating efficiency of individual normal single hematopoietic stem cells varied between samples. Eighty eight out of 192 single stem cells originated from cord blood grew into more than two cells, yielding plating efficiency of 45.8% and cells from the peripheral blood of two healthy donors 30.2% (58/192). In contrast, single LSC originated from the AML patients showed significantly lower plating efficiency with 14.6% (42/288), 3.6% (7/192) and 8.0% (23/288). These results directly confirmed quiescent and slowly dividing properties of LSC. In addition, plating efficiency of normal hematopoietic stem cells was shown to vary between their originating locations in healthy donors.