Isolation of Human CD34- Cells with High Aldehyde Dehydrogenase Activity Reveals a Novel Population with Hematopoietic Repopulating Potential.

Abstract

The successful development of stem cell-based therapies requires a thorough understanding of human hematopoietic stem cell (HSC) populations. Human CD34− cells engraft NOD/SCID mice with low efficiency by intravenous (IV) transplant. However, intra-femoral injection into immune deficient mice has identified potent human repopulating cells from CD34+ and CD34− subfractions. We recently described 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) activity. Hematopoietic progenitor function and in vivo reconstituting ability were exclusively maintained within the ALDHhiLin− population, which demonstrated variable expression of CD34. Here, we compared the repopulating ability of purified CD34+ALDHhiLin− and CD34−ALDHhiLin− populations to traditionally isolated CD34+Lin− and CD34−Lin− cells. Sorting of Lin− cells from human UCB isolated CD34−ALDHhi and CD34+ALDHhi cells (>96% purity) at an overall frequency of 4.4±1.3% or 29.1±3.5%, respectively. In contrast to CD34−Lin− cells, ALDHhiCD34−Lin− cells demonstrated robust clonogenic progenitor function in vitro (1 CFU in 9 cells, n=3), and total colony production was further increased in ALDHhiCD34+Lin− cells (1 CFU in 4.5 cells, n=4) (p<0.05). Human hematopoietic repopulation was consistently observed in the bone marrow (17.2±4.2%), spleen (0.8±0.2%), and peripheral blood (0.7±0.3%) of NOD/SCID β2M null mice 6–8 weeks after IV transplant with 103–104 purified ALDHhiCD34+Lin− cells (n=14). Similarly, intra-femoral injection (IF) of ALDHhiCD34+Lin− cells resulted in robust human repopulation (n=5). IV injection of equivalent doses of either ALDHhiCD34+Lin− or CD34+Lin− cells showed similar levels and frequencies of human hematopoietic engraftment. Repopulating ALDHhiCD34+Lin− cells also differentiated into cells expressing markers for mature myeloid (CD33, CD14), B-lymphoid (CD19, CD20) cells and primitive repopulating cells (CD34+CD38−) at similar frequencies as CD34+Lin− cells (n=5). IV injection of 2x104–1x105 ALDHhiCD34−Lin− cells engrafted at 0.2–0.3% in the BM of 3 of 4 NOD/SCID β2M null mice, whereas IV injection of up to 4x105 CD34−Lin− cells produced no detectable human engraftment (n=6). IF-injected ALDHhiCD34−Lin− cells engrafted the injected bone in 2 of 3 NOD/SCID mice at low levels and did not efficiently migrate to the non-injected femur or tibiae. In summary, the human UCB ALDHhiLin− population includes both CD34+ and CD34− cells capable of bone marrow homing and hematopoietic reconstitution. Therefore, isolation of CD34− cells based on high ALDH activity may reveal a novel population of hematopoietic stem and progenitor cells.