Human hematopoietic stem cells (HSC) are commonly purified by the phenotypic expression of cell surface markers such as CD34. We have recently characterized a novel strategy to purify reconstituting HSC from human umbilical cord blood (UCB) by lineage depletion (Lin) followed by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity. Lin cells with high ALDH activity (ALDHhiLin) represented approximately 0.1% of total UCB mononuclear cells and demonstrated enriched expression of the primitive HSC markers CD34 (91.0±2.9%) and CD133 (70.9±4.0%). Most notably, clonogenic progenitor function and in vivo reconstituting ability in immune deficient mice were exclusive to the ALDHhiLin population. Here, we have further purified the ALDHhiLin population based on the expression of CD133, or prominen, a non-restricted surface molecule expressed on primitive progenitor cells of hematopoietic, endothelial, and neural epithelial lineages. ALDHhiCD133 and ALDHhiCD133+ cells, sorted to >95% purity, represented 14.7±2.1% and 23.2±4.3% of the total human UCB Lin population respectively (n=6). Both ALDHhiCD133Lin and ALDHhiCD133+Lin cells demonstrated clonogenic progenitor function in vitro. However, total colony production was significantly enhanced (p<0.05) in ALDHhiCD133Lin cells (1 CFU in 3.5 cells, n=5) when directly compared to ALDHhiCD133+Lin cells (1 CFU in 10 cells, n=6). Human hematopoietic repopulation was consistently observed in the bone marrow, spleen, and peripheral blood of NOD/SCID (n=23) and NOD/SCID B2M null (n=27) mice transplanted with as few as 103 ALDHhiCD133+Lin cells, whereas transplantation of up to 2x105 ALDHhiCD133Lin cells produced no detectable human engraftment. BM repopulation at limiting dilution demonstrated increased NOD/SCID repopulating ability elicited by ALDHhiCD133+Lin cells when directly compared to CD133+Lin cells not selected for ALDH activity. Repopulating ALDHhiCD133+Lin cells differentiated into cells expressing markers for mature myeloid (CD33, CD14) and B-lymphoid (CD19, CD20) cells. ALDHhiCD133+Lin cells also supported the maintenance of primitive cell phenotypes up to 8 weeks post-transplantation (2.4±0.7% CD34+CD38, 5.5±0.6% CD34+CD133+, n=6) and the repopulating function of these cells are currently being confirmed by secondary transplantation. We are also investigating the ability of ALDHhiCD133+Lin cells to mediate tissue repair in non-hematopoietic organs. Fractionation of human HSC based on combined expression of CD133 and high ALDH activity provides a rigorous selection of purified hematopoietic stem and progenitor cells that maintain primitive characteristics after transplantation, and may be considered a potential alternative to CD34+ cell isolation.

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