Analysis of hematopoietic stem cells using a composite approach

Stem cells or Cancer stem cells (CSCs) have now been identified in different type of tissues by using surface markers. Functional assays such as ALDEFLUOR and side population which are marker independent have been additional approaches. However, whether all these approaches identify the same population of cells remain uncertain. To address this issue we have used hematopoietic stem cells as a model. Peripheral blood stem cells enumerated by CD34 are used routinely in bone marrow transplantation which supports the recovery of bone marrow after ablative chemotherapy or radiation. Hematopoietic stem cells (HSCs) were obtained from normal donor bone marrow (n = 5) and G-CSF stimulated peripheral blood stem cells (PBSCs) (n = 5) from patients undergoing leukapheresis prior to bone marrow transplantation. The stem cells were identified by combining CD34 expression with functional assays (ALDEFLUOR and side population). The cell cycle profile was further determined by simultaneous labeling of these cells with Hoechst and Pyronin Y. The simultaneous analysis showed that both CD34+ and CD34- cells co-exist with ALDH1A1+ cells but side population did not segregate with CD34+ cells. Though stem cell populations identified by functional assays were different, the cell cycle analysis showed that both ALDH1A1+ and CD34+ cells were in the G1 phase of cell cycle rather than in the quiescent (G0) phase.