Differential expansion of human primitive hematopoietic stem cell subsets: Correlation between phenotype, cell division history and frequency scid repopulating cells

Abstract

Listen

Translate article

CD34+ cells were isolated from G-CSF mobilized normal volunteers and cultured for 4 days in serum-free medium (X-VIVO 15) in the presence of SCF (20 ng/ml) + TPO (50 ng/ml) + FLT-3L (50 ng/ml). The phenotype (CD38, Thy-1, c-kit, HLA-DR) and cell division history (CFSE labeling) of CD34+ cells were examined. The frequency of LTC-IC and SCID repopulating cells (SRC) was measured by limiting dilution assays. After 4 days, the total number of CD34+ cells was maintained but a 25% reduction in the frequency of CFU and a 5-fold increase in LTC-IC were observed. The percentage of CD34+CD38− and CD34+DR− cells increased 57 and 5 fold, respectively, as > 85% of the CD38− and DR− cells divided 5 to 6 times from Day 0 to Day 4. The percentage of CD34+/c-kit+ was maintained after 4 days of culture even though > 90% of the cells had divided 2 to 5 times. In contrast, there was a 6-fold reduction in Thy-1+ cells while 60% of the Thy-1+ cells detected at Day 4 had undergone 0 to 2 divisions. Cultures of sorted CD34+Thy-1+ cells indicated that 3 or more cell divisions resulted in a gradual loss of Thy-1 expression. This data indicates that the combination of SCF+TPO+FLT-3L preferentially expands CD34+CD38− and DR− subsets. Data correlating CD34+ subsets, number of cell divisions and frequency of LTC-IC and SRC will be presented.