Umbilical cord blood (CB) CD34(+) cells, on the basis of flow cytometry analysis, are comprised of multiple populations. In in vitro assays, only CD34(regular) FSC(high) cells are functional and low percentages of nonfunctional CD34(regular) FSC(low) cells were determined to be present in liquid-stored CB. Liquid-stored CD34(regular) FSC(high) cells prior to cryopreservation were judged to be functional by the formation of erythroid and myeloid colonies and transmigration assays. We have further evaluated the occurrence of apoptosis in CB CD34(+) cells using various apoptotic markers to understand better the influence of storage conditions that could be utilized with transplantation of CB. Of the CD34(regular) FSC(low) cells shown in the present study, 20-45% were labeled with the apoptotic reagents annexin-V, fluorescent caspase peptide substrates, and the anti-mitochondrial antibody APO2.7, but these cells were minimally stained with 7-aminoactinomycin-D (7-AAD). These apoptotic reagents identify different cellular targets, indicating the initiation of the apoptotic cascade prior to cryopreservation/thawing. Following cryopreservation and thawing, the apoptotic markers SYTO-16, tetramethyl rhodamine ethyl ester (TMRE), and 7-AAD showed the presence of apoptotic cells. After cryopreservation/thawing, enumeration of CB CD34(+) cells was reduced 10-65% when excluding cells positive for apoptotic markers. We attempted to limit the progression of apoptosis observed after cryopreservation/thawing by the addition of anti-apoptotic reagents z-VAD-fmk (100 microM) and Q-VD-OPH (100 microM) (peptide inhibitors of caspases) without or with the inclusion of survival reagents for CD34(+) cells-stromal-derived factor-1 (SDF-1), stem cell factor (SCF), thrombopoietin, and diprotin A, an inhibitor of CD26 prior to cryopreservation. The expression of apoptosis markers was minimally affected even when using combinations of caspase inhibitors/ CD34(+) cell survival cytokines in an attempt to block apoptosis caused by cryopreservation/thawing. Decreases in apoptosis marker reactivity following cryopreservation were not observed except for a reduced expression of APO2.7 reactivity with z-VAD-fmk and Q-VD-OPH caspase inhibitors. The ability of the inhibitors of apoptosis of CD34(+) cells to generate CFU-GM, CFU-MK, or BFUE colonies was also unaffected except with z-VAD-fmk (100 microM) and Q-VD-OPH (100 microM). The occurrence of apoptosis, as measured by flow cytometry with selected apoptotic markers, suggests a reduction in the number of viable CD34(+) cells.