In the absence of conclusive assays capable of determining the functionality of ex vivo expanded human hematopoietic progenitor cells, we combined cell tracking with the membrane dye PKH2, immunostaining for CD34, and limiting dilution analysis to estimate the frequency of long-term hematopoietic culture-initiating cells (LTHC-ICs) among de novo-generated CD34+ cells. Umbilical cord blood (CB) and bone marrow (BM) CD34+ cells were stained with PKH2 on day 0 and cultured with stem cell factor (SCF) and interleukin-3 (IL-3) in short-term stromal cell-free suspension cultures. Proliferation of CD34+ cells in culture was tracked through their PKH2 fluorescence relative to day 0 and the continued expression of CD34. As such, it was possible to identify cells that had divided while maintaining the expression of CD34 (CD34+PKH2dim) and others that expressed CD34 but had not divided (CD34+PKH2bright). In all such cultures, a fraction of both BM and CB CD34+ cells failed to divide in response to cytokines and persisted in culture for up to 10 days as CD34+PKH2bright cells. Between days 5 and 7 of culture, CD34+PKH2bright and CD34+PKH2dim cells were sorted in a limiting dilution scheme into 96-well plates prepared with medium, SCF, IL-3, IL-6, granulocyte-macrophage colony-stimulating factor, and erythropoietin. Cells proliferating in individual wells were assayed 2 weeks later for their content of clonogenic progenitors and the percentage of negative wells was used to calculate the frequency of LTHC-ICs in each population. Among fresh isolated BM and CB CD34+ cells, the frequencies of LTHC-ICs were 2.01% +/- 0.98% (mean +/- SEM) and 7.56% +/- 2.48%, respectively. After 5 to 7 days in culture, 3.00% +/- 0.56% of ex vivo-expanded BM CD34+PKH2bright cells and 4.46% +/- 1.10% of CD34+PKH2dim cells were LTHC-ICs. In contrast, the frequency of LTHC-IC in ex vivo expanded CB CD34+ cells declined drastically, such that only 3.87% +/- 2.06% of PKH2bright and 2.29% +/- 1.75% of PKH2dim cells were determined to be initiating cells after 5 to 7 days in culture. However, when combined with a calculation of the net change in the number of CD34+ cells in culture, the sum total of LTHC-ICs in both BM and CB cells declined in comparison to fresh isolated cells, albeit to a different degree between the two tissues.(ABSTRACT TRUNCATED AT 400 WORDS)