Dissociation between stem cell phenotype and NOD/SCID repopulating activity in human peripheral blood CD34+ cells after ex vivo expansion

Objectives The relationship between phenotype and function in ex vivo–cultured human hematopoietic stem cells (HSC) remains poorly understood. We investigated the effects of a short-term serum-free culture on the relationship between stem cell phenotype, cell division history, and function in human CD34+ cells. Methods G-CSF–mobilized peripheral CD34+ cells were cultured for 4 days with stem cell factor, flt-3 ligand, and thrombopoietin. The phenotype (CD34, CD38, HLA-DR, c-kit), cell division history, colony-forming cell (CFC), long-term culture-initiating cell (LTC-IC), and NOD/SCID repopulating activities were evaluated at Day 0 and 4. Results We observed a loss of CD38, HLA-DR, and c-kit surface expression resulting in a drastic increase in CD34+CD38−, CD34+HLA-DR−, and CD34+c-kit−/low cells at Day 4. In contrast, the frequency of Thy-1+ cells was maintained. We observed a 1.3-fold expansion of CFC, a 4.8-fold increase in LTC-IC, and an overall maintenance of the NOD/SCID repopulating cell activity. CD34+CD38− and CD34+HLA-DR− cells detected at Day 4 displayed the most active pattern of division (4 to 5 divisions) whereas 60% of CD34+Thy-1+ cells divided 0 to 2 times during the same period. At Day 4, the NOD/SCID repopulating activity was associated with Thy-1+ cells with no more than 2 divisions. Conclusions Our results show that the relationship between stem cell phenotype and function is dramatically altered in cultured CD34+ cells. Thy-1 expression and cell division history appear to be superior to CD38, HLA-DR, and c-kit, or to homing molecules (CXCR4, VLA-4) as predictors of the repopulating activity of cultured peripheral CD34+ cells.