The Enhancement of Short-Term Engraftment in NOD/Scid Mice after Double Cord Blood Transplantation Is Mediated by CD34+ Cells

Abstract

Introduction: Cord blood (CB) is an attractive source of hematopoietic stem cells, however, the low number of cells available hampers its use in adult patients. Transplantation of two unrelated CB units seems to overcome the cell dose limitations for adult patients with at least similar engraftment levels compared to single CB transplantation with sufficient cell numbers. Pre-clinical studies show an important role for total cell numbers in the graft, but also CD34+ stem cells or CD34− cells, including NK cells and/or Lin+, CD3+ T cells may play a facilitating role, possibly via a graft-versus-graft effect. In this study we explored the role of the CD34+ and CD34− cells in facilitating engraftment in NOD/Scid mice after double CB transplantation.Methods: Cohorts (ch.) of irradiated (3.5Gy) NOD/Scid mice (n=9–18 mice per cohort; 3 independent experiments) were transplanted with a combination of 1.105 CD34+ cells (MACS purified; >94% pure) and 1.107 CD34- cells derived from one CB unit. In addition, mice received a combination of CD34+ and CD34− cells, or CD34+, or CD34− cells only, derived from a second CB unit (Table 1). BM engraftment was assessed by the percentage of human CD45+ cells at 6 weeks after transplantation. In addition, the percentage of human lymphoid (CD19/CD3) and myeloid (CD33) cells in the BM were evaluated at 6 weeks. Human platelet (hPLT) and human CD45+ cell recovery in blood was measured from 3 to 6 weeks after transplantation.Results: At 6 weeks, 67% of the mice in the single CB cohort (ch.1), 75% in the cohort co-transplanted with CD34− cells (ch.4), 83% in the cohort co-transplanted with CD34+ cells (ch.3) and 89% in the cohort co-transplanted with the combination of CD34+ and CD34− cells of the second donor, showed engraftment (>10% human CD45+ cells) in the BM. Mice that were co-transplanted with CD34+ cells only (ch.3) had a significantly higher level of engraftment in the BM than mice receiving no second CB unit (ch.1) (54% vs. 22%; p=0.011). A similar engraftment enhancing effect was observed in mice co-transplanted with a combination of CD34+ and CD34− cells (ch.2) (50% p=0.016 vs. ch.1). In contrast, no significant enhancing effect was observed after co-transplantation of CD34− cells only (ch.4) (36%). The distribution between human lymphoid and myeloid cells in BM was similar between al cohorts. All engrafted mice had sustained mixed chimerism in the BM without pre-dominance of a single CB unit during the time of evaluation. hPLT recovery was significantly increased in mice co-transplanted with CD34+ cells, either alone (ch.3; 607plt/μl) or in combination with CD34− cells (ch.2; 1076 plt/μl) compared to mice receiving no second CB (ch.1; 183 plt/μl) (p=0.04 and p<0.001 resp.). The observed mean hPTL concentration in cohort-2 and -3 was greater than expected by the sum of the single CBs (305 plt/μl), indicating a synergistic effect. In mice that were cotransplantated with CD34− cells from a second donor (ch.4) no significant effect on hPLT recovery (253 plt/μl) was observed. Similar results were obtained for hCD45 recovery in peripheral blood.Conclusion: These results show that the engraftment facilitating effect of co-transplantation of CD34+ cells only are similar compared to co-stransplantation of both CD34+ and CD34− cells. Therefore, it is suggested that enhancement of short-term engraftment in NOD/Scid mice by double cord blood transplantation is mainly mediated by the CD34+ cells in the second CB.Table 1: Composition of the transplants per cohort.Cohort-1 received a single CB transplantation of donor-1 or donor-2. Cohort-2 received a normal double CB transplantation.CohortTransplanted cells of donor-1Co-transplanted cells of donor 21CD34+ + CD34−ORCD34+ + CD34−2CD34+ + CD34−ANDCD34+ + CD34−3CD34+ + CD34−ANDCD34+4CD34+ + CD34−ANDCD34−