898. Human Hematopoietic Repopulating Stem and Progenitor Cells Can Be Efficiently Selected Based on High Aldehyde Dehydrogenase Activity

Abstract

Top of pageAbstract Human hematopoietic stem cells (HSC) are commonly purified by the expression of cell surface markers, such as CD34. Since cell phenotype can be altered by cell cycle progression or ex vivo culture, purification based on conserved stem cell function may represent a more reliable way to isolate stem cell populations. Human hematopoietic progenitor cells express increased levels of cytosolic aldehyde dehydrogenase (ALDH), an enzyme responsible for the resistance of HSC to alkylating agents such as cyclophosphamide. We purified primitive HSC from human umbilical cord blood (UCB) by lineage depletion (Lin-) followed by the selection of cells with high ALDH activity using the fluorescent substrate Aldefluor™. Side scatter low, ALDH high (ALDHhi) cells comprised 22.6 ± 3.0% of the Lin- population (n=9), and represented 0.1 ± 0.01% of total MNC from UCB (n=5). In comparison to ALDHloLin- cells, the ALDHhiLin- population demonstrated significantly (p < 0.05) enriched expression of the pan-leukocyte marker CD45 (98.6 ± 0.7%), the primitive cell surface markers CD34 (91.0 ± 2.9%), CD133 (70.9 ± 4.2%), CD117 (83.1 ± 2.7%), and platelet-derived endothelial cell adhesion molecule (PECAM-1) or CD31 (97.9 ± 0.8%). ALDHhiLin- cells were also highly enriched for primitive HSC markers CD34 +CD38− (25.5 ± 3.7% vs 5.1 ± 1.3%, p < 0.005), and CD34 + CD133+ (72.7 ± 4.0 vs 12.0 ± 3.1, p < 0.00005), cell phenotypes associated with enhanced repopulating function in vivo. Human hematopoietic progenitor function was enriched in the ALDHhiLin- population (1 CFU in 4.4 cells, n=5), while the ALDHloLin- population had little in vitro colony forming ability at cell densities up to 5 × 104 cells. Multilineage human hematopoietic repopulation was consistently observed in the bone marrow, spleen and peripheral blood of immune deficient mice transplanted with ALDHhiLin- cells, whereas ALDHloLin- cells produced no detectable human engraftment. Direct comparison of repopulation using the NOD/SCID (n=37) and NOD/SCID beta-2 microglobulin (B2M) null (n=41) models demonstrated that 10-fold greater numbers of ALDHhiLin- cells were needed to engraft the NOD/SCID mouse as compared to the more permissive NOD/SCID B2M null mouse, suggesting that the ALDHhiLin- population contained committed progenitors as well as primitive SCID repopulating cells. Engrafting ALDHhiLin- cells in the mouse bone marrow differentiated into cells that express lineage markers for mature myeloid (CD33), mature B-lymphoid (CD19) and primitive repopulating (CD34 + CD38−) cells. We are currently evaluating the ALDHhiLin- population as a novel target for retroviral and lentiviral gene therapy applications. Cell fractionation based on lineage depletion and ALDH activity provides a reproducible method to purify human stem and progenitor cells based primarily on stem cell function rather than hematopoietic cell surface markers.