Evaluation of ex vivo expansion potential of cord blood and bone marrow hematopoietic progenitor cells using cell tracking and limiting dilution analysis

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⁺PKH2(dim)) and others that expressed CD34 but had not divided (CD34⁺PKH2(bright)). 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⁺PKH2(bright) cells. Between days 5 and 7 of culture, CD34⁺PKH2(bright) and CD34⁺PKH2(dim) 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⁺PKH2(bright) cells and 4.46% ± 1.10% of CD34⁺PKH2(dim) 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 PKH2(bright) and 2.29% ± 1.75% of PKH2(dim) 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. These data suggest that the number of LTHC-ICs declines among de novo-generated CD34⁺ cells in culture and that for a relatively long period of time a substantial number of LTHC-ICs remain unresponsive to cytokine stimulation. Furthermore, these studies demonstrate the utility of this approach in investigating the hematopoietic potential of ex vivo-expanded progenitor cells and may provide a simple in vitro assay for the assessment of engraftment potential of hematopoietic tissues.