Cord Blood (CB) CD34⁺ and CD3⁺ Cells with Decreasing Levels of Their Mitochondrial Potential Are Not Detectable with 7-AAD, Have Impaired Function and Ultrastructural Morphology Characteristic of Early Apoptosis.

Background: Banked CB is an effective source of hematopoietic stem cells for allogeneic bone marrow reconstitution. Critical indices of engraftment potential and quality of a CB graft are the nucleated cell dose, CD34⁺ cell content and total CFU. Determination of CD34⁺ cell viability by flow cytometry depends on 7-actinomicin D (7-AAD) exclusion, but its diffusion into cells depends on permeability of the cell membrane. Earlier stages of the death pathway (apoptosis) escape detection by 7-AAD. Aims of our study:

• to establish a sensitive method for detection of early apoptosis in CB cells and to evaluate their ultrastructural morphology,

• to determine the extent to which early apoptosis may disclose reduced functionality of CD34⁺ and CD3⁺ cells and therefore, whether early apoptosis should be investigated routinely in CB grafts before transplantation and

• to investigate the presence and degree of apoptosis in CD34⁺ and CD3⁺ cells upon CB storage at RT and after cryopreservation/thawing, as well as the effect of post-thaw DMSO removal.

Results: After CB storage over 48 hrs, evaluation by multiparameter flow cytometry with the mitochondrial potential (MP) marker DILC, Annexin V, 7-AAD and surface cell markers, identified 3 populations with decreasing MP and negative 7-AAD: 1. DILC with high MP (MP^High)/Annexin(−); 2. DILC with intermediate MP (MP^Interm)/Annexin(−) and 3. MP^Interm/Annexin(+). Groups were sorted for CD3⁺ and CD34⁺ cells, ultrastructural morphology was evaluated by electron microscopy (EM) and their function by culture with Phytohemaglutinin/IL-2 (PHA) for CD3⁺ and by CFU assay for CD34⁺ cells. EM revealed lymphocyte shrinkage, nuclei with condensed chromatin and polarized clusters of organelles, findings suggestive of early stages of apoptosis. In addition, CD3⁺ cells with MP^High had higher stimulation than those with MP^Interm and impaired more when cells became Annexin(+) (ratio MP^High/MP^Interm=54; p<0.001). Furthermore, CD34⁺ cells with MP^High showed higher CFU than those with MP^Interm/Annexin(−) and those with Annexin(+), (CFU ratio MP^High/MP^Interm= 17.7 and 35.6 respectively, p<0.05). Evaluation of CD3⁺ and CD34⁺ cells upon CB storage over 48hrs and after cryopreservation/thawing showed decreased CD3⁺ cell viability (p<0.001). 7-AAD failed to detect cells under apoptosis (range 1.1–6.4%). We observed individual variations of the retention of function after cryopreservation/thawing of CD3⁺ cells (range 37–79%). In contrast, there were no significant variations in viability or function of CD34⁺ cells after cryopreservation/thawing. Thawed cells with DMSO dilution down to 2% concentration or DMSO removal (Dextran/Albumin wash) effected no significant change in viability or function.

Summary:

1. CB cells with decreased mitochondrial potential have impaired function and morphological characteristics of early apoptosis;

2. A combination of 7-AAD with a marker of mitochondrial potential (DILC) allows for the detection of early apoptotic cells which are not detected by 7-AAD;

3. CB-lymphocytes have immature appearance by ultrastructural morphology compared to those of adult peripheral blood;

4. Viability and function of CB-CD34⁺ cells were not significantly reduced by cryopreservation/thawing but those of CD3 cells were.

Lower viability and decreased function of CB-CD3 cells may affect the immunological interactions in double or multiple CB transplants as well as clonal expansion post-transplant.