[In Vitro Identification and Characteristics of CD34^- Leukemia Stem Cell in t(8;21) Acute Myeloid Leukemia]

To preliminarily identify the existence of CD34^- leukemia stem cell (LSC) in t(8;21) acute myeloid leukemia (AML) by in vitro test.

Bone marrow samples collected from newly diagnosed t(8;21) AML patients were tested. Lin^-CD34⁺ CD38^-(abbreviation, CD34⁺CD38^-), Lin^-CD34⁺CD38⁺ (abbreviation, CD34⁺CD38⁺) and Lin^-CD34^-CD38^-CD45^dimSSC^low(abbreviation, CD34^-"LSC") cell fractions were gated by flow cytometry after staining with fluorescent antibodies. Cells in G₀ phase were identified through Hoechst 33342 and pyronin Y staining. Aldefluor reagent was used to test aldehyde dehydrogenase (ALDH) activity. The above-mentioned 3 cell fractions were sorted, and mRNA levels of AML1-ETO and WT1 were measured by real-time quantitative PCR.

The 3 tested samples displayed the same tendency in ratio of the cells in G₀ phase: CD34^-"LSC">CD34⁺ CD38^->CD34⁺CD38⁺. The paired t-test of 53 patients showed that frequency of ALDH^br cells of both CD34⁺CD38^- and CD34^-"LSC" cell fractions was significantly higher than that of CD34⁺CD38⁺ (P<0.01), furthermore, the ALDH^br cell frequency was significantly higher in CD34^-"LSC" than that in CD34⁺ CD38^- (P<0.01). AML1-ETO mRNA levels of cells sorted from 3 patients were similar among the 3 cell fractions within each patient, whereas WT1 mRNA levels were significantly higher in CD34^-"LSC" than that in other 2 cell fractions.

CD34^- LSC may exist in t(8;21) AML, and may be more primitive than CD34⁺ LSC. These results promote the necessity to perform in vivo xenogeneic transplantation mice.