Recent studies suggest that the population of malignant cells found in human acute myelogenous leukemia (AML) arises from a rare population of leukemic stem cells (LSCs). LSCs have been documented for nearly all AML subtypes and have been phenotypically described as CD34 + /CD38 − or CD34 + /HLA-DR − . Given the potentially critical role of these primitive cells in perpetuating leukemic disease, we sought to further investigate their molecular and cellular characteristics. Flow cytometric studies using primary AML tissue showed that the interleukin-3 receptor alpha chain (IL-3R␣ or CD123) was strongly expressed in CD34 + /CD38 − cells (98 ± 2% positive) from 16 of 18 primary specimens. Conversely, normal bone marrow derived CD34 + /CD38 − cells showed virtually no detectable expression of the CD123 antigen. To assess the functional role of IL-3R␣ positive cells, purified CD34 + /CD123 + leukemia cells were transplanted into immune deficient NOD/SCID mice. These experiments showed that CD123 + cells were competent to establish and maintain leukemic populations in vivo. To begin to elucidate a biological role for CD123 in leukemia, primary AML samples were analyzed with respect to signal transduction activity in the MAPK, Akt, and Stat5 pathways. Phosphorylation was not detected in response to IL-3 stimulation, thereby suggesting CD123 is not active in conventional IL-3-mediated signaling. Collectively, these data indicate that CD123 represents a unique marker for primitive leukemic stem cells. Given the strong expression of this receptor on LSCs, we propose that targeting of CD123 may be a promising strategy for the preferential ablation of AML cells.

MRI has been employed to elucidate the migratory behavior of stem/progenitor cells noninvasively in vivo with traditional proton (1H) imaging of iron oxide nanoparticle-labeled cells. Alternatively, we demonstrate that fluorine (19F) MRI of cells labeled with different types of liquid perfluorocarbon (PFC) nanoparticles produces unique and sensitive cell markers distinct from any tissue background signal. To define the utility for cell tracking, mononuclear cells harvested from human umbilical cord blood were grown under proendothelial conditions and labeled with nanoparticles composed of two distinct PFC cores (perfluorooctylbromide and perfluoro-15-crown-5 ether). The sensitivity for detecting and imaging labeled cells was defined on 11.7T (research) and 1.5T (clinical) scanners. Stem/progenitor cells (CD34+ CD133+ CD31+) readily internalized PFC nanoparticles without aid of adjunctive labeling techniques, and cells remained functional in vivo. PFC-labeled cells exhibited distinct 19F signals and were readily detected after both local and intravenous injection. PFC nanoparticles provide an unequivocal and unique signature for stem/progenitor cells, enable spatial cell localization with 19F MRI, and permit quantification and detection of multiple fluorine signatures via 19F MR spectroscopy. This method should facilitate longitudinal investigation of cellular events in vivo for multiple cell types simultaneously.

IntroductionConventionally, human hematopoietic stem cells (HSCs) have been purified on the basis of the expression of cell surface molecules such as CD34 and CD133. [1][2][3][4][5] Cells expressing CD34 are capable of long-term hematopoietic reconstitution in immune-deficient mice and fetal sheep, [1][2][3]6,7 and most clinical protocols involving gene transfer, purified stem cell transplantation, and stem cell expansion have been designed with the use of CD34 ϩ populations. [8][9][10] However, findings in a variety of mammalian systems suggest that the human hematopoietic stem cell compartment is heterogeneous, including an array of cell phenotypes that exhibit repopulating function. [11][12][13][14] In fact, human CD34 ϩ and CD34 Ϫ populations possess repopulating ability, and CD34 expression is reversible in mouse and human cells. [15][16][17][18][19] Although CD34 selection has proven extremely useful for the isolation and characterization of human cells with enhanced repopulating function, there are several limitations to purifying hematopoietic cells based solely on cell surface phenotype. First, cell surface markers can vary between species and stem cell source. Murine long-term repopulating cells have been purified to a single cell with the use of CD34 Ϫ , c-kit ϩ , Sca-1 ϩ , Lin Ϫ cell markers, 11 whereas identification of a human single cell phenotype capable of repopulating murine or fetal sheep xenograft models remains elusive. 1,12,14 Second, human stem cell phenotype can be altered by cell cycle progression, 20,21 ex vivo culture, 14,19,22 and transplantation in vivo, 12,15,16 indicating a dissociation between hematopoietic cell phenotype and repopulating cell function. Third, the phenotype of transplanted stem cells may remain constant despite reduced functional activity. Cytokinemobilized peripheral blood cells demonstrate increased CD34 expression without an increase in nonobese diabetic/severe combined immunodeficiency (NOD/SCID) repopulating ability. 23,24 Finally, purification of human stem cells on the basis of hematopoietic markers may select cells with restricted developmental potential and may exclude cells with alternate stem cell or progenitor functions. Thus, methods to efficiently isolate human stem cells without relying entirely on phenotypic cell surface molecules are desirable.One promising strategy is HSC isolation according to a conserved stem cell function rather than phenotype. In the murine system, lymphohematopoietic stem cells have been isolated according to the high expression of the detoxifying enzyme aldehyde dehydrogenase (ALDH). [25][26][27] Cytosolic ALDH is also expressed at high levels in human hematopoietic progenitors, 28 (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-5-proprionic acid) fluorochrome that is metabolized by ALDH to an aminoacetate anion, retained within the cell because of its negative charge. Thus, the amount of fluorescent product that accumulates in viable cells correlates to ALDH activity, and cells with high ALDH activity can be selected from...