1031. Purification of Cells with High Aldehyde Dehydrogenase Activity Reveals a CD133 Negative Population with Enhanced Repopulating Ability after Ex Vivo Culture

Purified hematopoietic stem cell (HSC) populations selected as targets for gene therapy are required to efficiently incorporate viral gene products and retain the ability to reconstitute hematopoiesis after transplantation. Currently, HSC for gene therapy procedures are most commonly selected using CD34 expression. As an alternate strategy to purify cells based on a conserved stem cell function rather than phenotype, we have purified HSC from human umbilical cord blood (UCB) by lineage depletion (Lin-) followed by selection of cells with high aldehyde dehydrogenase (ALDH) enzyme activity. Hematopoietic progenitor function and in vivo reconstituting ability in immune deficient mice were exclusive to the ALDHhiLin|[minus]| population (Hess, 2004). Here, we have further purified the ALDHhiLin- population based on the expression of CD133, a cell surface protein expressed on primitive progenitor cells of hematopoietic, endothelial, and neural epithelial lineages. ALDHhiCD133|[minus]| and ALDHhiCD133+ cells (>95% purity), represented 14.7|[plusmn]|2.1% and 23.2|[plusmn]|4.3% of the human UCB Lin- population respectively (n=6). These subsets were primarily (75|[ndash]|95%) CD34+, and included a small population of CD34-CD38-Lin- cells, a phenotype associated with NOD/SCID repopulating cell (SRC) function. In 4 day serum free culture, both ALDHhiCD133-Lin- and ALDHhiCD133+Lin- cells expanded 2.5 fold, and retained CD34 expression at approximately 25% and 45% respectively (n=4). Both ALDHhiCD133-Lin- and ALDHhiCD133+Lin- populations demonstrated clonogenic progenitor function in vitro. However, human hematopoietic repopulation was observed in the bone marrow (BM), spleen, and peripheral blood of NOD/SCID (n=23) and NOD/SCID B2M null (n=27) mice transplanted with as few as 102 ALDHhiCD133+Lin- cells. Transplantation of up to 2|[times]|105 ALDHhiCD133-Lin- cells did not produce human engraftment. Limiting dilution analysis in the NOD/SCID B2M null model showed a frequency of 1 SRC in 485 ALDHhiCD133+Lin- cells. Transplanted ALDHhiCD133-Lin- cell progeny expressed markers for mature myeloid and B-lymphoid cells and supported the maintenance of primitive cell phenotypes (34.3|[plusmn]|5.3% CD34+) 8 weeks after transplantation. Long-term repopulating function of these cells was confirmed by successful engraftment in secondary recipients (n=5). ALDHhiCD133+Lin- cells cultured for 4 days ex vivo and transplanted at 1-2|[times]|105 cells engrafted NOD/SCID mice at 2.1|[plusmn]|0.8% (n=9). Remarkably, 1-2|[times]|105 cultured cells derived from the purified ALDHhiCD133-Lin- population also reconstituted in NOD/SCID mice (6.7|[plusmn]|2.4%, n=5), demonstrating a gain of SRC function after ex vivo culture of ALDHhiCD133-Lin- cells. Fractionation of human UCB cells based on high ALDH activity and CD133 expression provides a rigorous selection of human HSC that maintained primitive cell phenotype after transplanation, possessed repopulating function in secondary recipients, and retained repopulating potential after culture. These unique characteristics indicate that ALDHhiLIn- populations may be superior to CD34+ cell selection for clinical gene therapy applications.