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

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 ALDH^hiLin⁻ population, which demonstrated variable expression of CD34. Here, we compared the repopulating ability of purified CD34⁺ALDH^hiLin⁻ and CD34⁻ALDH^hiLin⁻ populations to traditionally isolated CD34⁺Lin⁻ and CD34⁻Lin⁻ cells. Sorting of Lin⁻ cells from human UCB isolated CD34⁻ALDH^hi and CD34⁺ALDH^hi cells (>96% purity) at an overall frequency of 4.4±1.3% or 29.1±3.5%, respectively. In contrast to CD34⁻Lin⁻ cells, ALDH^hiCD34⁻Lin⁻ cells demonstrated robust clonogenic progenitor function in vitro (1 CFU in 9 cells, n=3), and total colony production was further increased in ALDH^hiCD34⁺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 ALDH^hiCD34⁺Lin⁻ cells (n=14). Similarly, intra-femoral injection (IF) of ALDH^hiCD34⁺Lin⁻ cells resulted in robust human repopulation (n=5). IV injection of equivalent doses of either ALDH^hiCD34⁺Lin⁻ or CD34⁺Lin⁻ cells showed similar levels and frequencies of human hematopoietic engraftment. Repopulating ALDH^hiCD34⁺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 ALDH^hiCD34⁻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 ALDH^hiCD34⁻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 ALDH^hiLin⁻ 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.