With the increase in use of umbilical cord blood (UCB) as a source of haematopoietic stem cells (HSC), the qualitative screening of HSC function in cryopreserved UCB has become an issue. While studies have shown the importance of total nucleated cell (TNC) numbers (Rubinstein et al, 1998; Rocha et al, 2000) or CD34+ cell contents, as a marker to predict haematopoietic potential (Wagner et al, 2002; Aroviita et al, 2003), it has been reported that CD34+ cells in UCB or mobilised peripheral blood could often undergo early apoptosis (Schuurhuis et al, 2001; de Boer et al, 2002; Mastino et al, 2003). Therefore, we investigated the possibility that the early apoptosis of CD34+ cells occurs in cryopreserved UCB, and that it could be an important factor in the functional heterogeneity of UCBs.
The increased use of umbilical cord blood (UCB) raises issues regarding the quality of cryopreserved UCB. This study investigated whether early apoptosis of CD34+ cells is part of the functional heterogeneity of cryopreserved UCB. Annexin V binding of CD34+PI(−) cells showed wide variations in both fresh and cryopreserved UCBs, with greater variation among units frozen for >5 years. Xenotransplantation of sorted cells into non-obese diabetic severe combined immunodeficient mice demonstrated that the Annexin V assay identified most repopulating activities in UCB units. Thus, early apoptosis of CD34+ cells could influence the outcome of transplantation using cryopreserved UCB.
Materials and methods
UCB collection and cell purification
Frozen UCB not destined for clinical use due to lack of human leucocyte antigen (HLA) information were used. UCB samples were either frozen after red blood cell fractionation by controlled-rate freezing, or frozen as mononuclear cells (MNC). Aliquots of fresh UCB samples were used for screening under informed consent according to the Institutional Bioethics Review Board of the Catholic University of Korea. MNC were separated using density gradient (<1·077) centrifugation and resuspended in Ca++/Mg++ free Hank's balanced salt solution containing 2% fetal bovine serum for further assay. CD34+ cells were purified using a CD34 progenitor cell selection system (DynalBiotech, Oslo, Norway).
Flow cytometric analysis for Annexin V binding
Mononuclear cells were stained with anti-CD34-allophycocyanin (APC) (BD Pharmingen, San Diego, CA, USA), washed, incubated for 30 min with Annexin V-fluorescein isothiocyanate (FITC) (BD Pharmingen) in the binding buffer, then stained with propidium iodide (PI: 1μg/ml) for flow cytometry. Aliquots stained with isotype control antibodies labelled with equivalent fluorochromes were used to set the control gate excluding 99·9% of PI(−) cells.
Xenotransplantation into non-obese diabetic severe combined immunodeficient (NOD/SCID) mice
Transplantation of human UCB cells into NOD/SCID mice (Shultz et al, 1995) was performed as described (Kim et al, 2004). Engraftment of human cells in these mice was analysed by flowcytometry using anti-human CD45-phycoerythrin (PE), CD71-PE, CD19, 20-PE, or CD13, 15-PE antibodies (BD Pharmingen) in the presence of 5% human serum and 2·4G2 (anti-mouse Fc receptor antibody).
Statistical differences between groups were analysed using Student's t-test. P-values <0·05 were considered significant.
Extensive heterogeneity in early apoptosis of CD34+ cells from cryopreserved UCB
Umbilical cord blood samples that had been cryopreserved for various periods were screened for Annexin V(+) binding (Vermes et al, 1995) to CD34+PI(−) populations (Fig 1A). Specificity of the assay was confirmed by <2% Annexin V(+) binding in freshly collected peripheral blood cells. Extensive variations, ranging from 10% to 44% (mean 30% ± 11%), were found in Annexin V(+) binding to CD34+PI(−) cells in 11 UCB units cryopreserved for <1 year (Fig 1B). Thirteen units cryopreserved for 1–3 years exhibited comparable extents of heterogeneity with a similar degree of Annexin V(+) binding (32% ± 11%). The percentages of Annexin V(+) binding were comparable for units frozen after RBC fractionation or those frozen after MNC separation. Interestingly, greater variation in Annexin V(+) binding of CD34+PI(−) cells was observed between 16 units that had been cryopreserved for 5–7 years (25–73%), with a higher mean % of Annexin V(+) cells (52% ± 15%) (Fig 1B).
To determine whether this early apoptosis could be attributed to cryopreservation, 56 units of fresh, unfrozen UCB were similarly screened (Fig 1C). Surprisingly, these fresh UCBs exhibited comparable levels of early apoptosis to units cryopreserved for <3 years. Notably, the percentage of Annexin V(+) cells in the CD34+PI(−) fraction, but not in MNCs, were significantly lower in UCB units separated less than 6 h after collection, than those separated after 6–24 h (25% vs. 35%, P = 0·004) (Fig 1C). These results show that both fresh and frozen UCBs exhibit extensive variations in the early apoptosis of CD34+ cells, with greater variations among units subjected to long-term storage.
Functional significance of the Annexin V assay for subsequent engraftment of UCB cells
To examine the functional implications for this screening, we examined the extent to which CD34+PI(−) Annexin V(−) cells could exclusively represent the repopulating potential of HSCs in a given UCB. CD34+PI(−) cells were sorted into fractions with different Annexin V staining intensities (Fig 2A), and transplanted into sublethally irradiated NOD/SCID mice. The Annexin V(−) population exhibited the greatest repopulating activity in engraftment (70% ± 9%, Fig 2B), whereas equivalent numbers of CD34+ cells from the Annexin Vlow or Annexin Vhigh fraction exhibited only basal levels of engraftment (6% ± 3% and 0·5% ± 0·4%; P = 0·0006 and 0·0004 respectively). However, no additional heterogeneity in repopulating activity was observed among the Annexin V(−) cells, as further sorting of the populations into fractions with different intensities (Fig 2C) did not result in differences in engraftment levels (Fig 2D) or lympho-myeloid differentiation of engrafted cells (Fig 2E). These results demonstrated that the Annexin V binding assay is selective, but sufficient to screen most of the effective repopulating activities contained in a given UCB sample.
Interest in screening for quality of cryopreserved UCBs has recently increased, as reflected by the recent recommendation to use the cells in the tube segment of the freezing bag as a final quality control before transplantation (Rodriguez et al, 2005). The present study showed extensive variations in the extent of early apoptosis among CD34+ cells of cryopreserved UCB. Further, xenotransplantation studies showed the functional relevance of this assay to effective repopulating activities in UCBs, supporting the benefits of additional screening on UCBs. In particular, the assay could be relevant for the selection of long-term frozen (>5 years) UCB units with substantial variations in early apoptosis, the recruitment of which become more feasible with the recent demonstration of NOD/SCID repopulating activities using 15-year-frozen UCB samples (Broxmeyer et al, 2003).
In summary, these findings suggest that early apoptosis of CD34+ cell could be an important but previously unrecognised parameter in cryopreserved UCB, and that an additional assay for this parameter could enable the more efficient selection and utilisation of cryopreserved UCBs to improve outcomes of UCB transplantation.
Current study was supported by a grant for a high-performance cell therapy project (0405-DB01-0104-0006) from the Ministry of Health and Welfare, Republic of Korea, and in part by a NITR/Korea FDA grant for Biologics Evaluation Research.