Delayed Platelet Recovery Following Cord Blood Transplantation: Insights from a Mouse Model.

Umbilical cord blood (CB) is an important source of hematopoietic stem cells for stem cell transplantation and is being used with increasing frequency. A major concern related to clinical CB transplantation is the long delay in platelet recovery. Megakaryopoiesis is characterized by the acquisition of lineage-specific markers (e.g. CD41) during the early stages that is followed by polyploidization (DNA content > 4N) during the later stages of megakaryopoiesis. Using a newborn blood (NB) model of CB transplantation that was previously developed in our laboratory, we asked whether the delay in platelet recovery is a result of a decrease in the rate of megakaryocyte production or a delay in their maturation. C57BL/6 mice were transplanted with either murine adult bone marrow (BM) cells or murine new-born blood (NB) cells following lethal irradiation. We had previously shown that the concentration of Lin-Sca-1+c-Kit+ stem cells in murine adult BM was approximately 3 times higher than that in NB. To correct for this difference in stem cell concentration, irradiated mice were transplanted with either 0.5 ×106 BM cells or 2×106 NB cells. Platelet counts at 2 and 4 weeks were lower in mice transplanted with NB cells than in mice transplanted with BM cells (Table 1). Interestingly, the platelet counts became comparable in NB and BM recipients at 8 weeks post-transplantation. We compared the ability of BM cells from both NB and BM recipients to form CFU-Meg colonies in methylcellulose. At 2 and 4 weeks post-transplantation, BM cultures derived from NB recipients generated fewer CFU-Meg colonies than cultures from BM recipients (Table 1). Interestingly, after 8 weeks, the numbers of CFU-Meg from both stem cell sources were similar. We also compared the ability of BM cells from NB and BM recipients to differentiate into megakaryocytes in liquid culture, using CD41 expression and polyploidy as markers of megakaryocytic maturation. At 2 weeks post-transplantation, cultures from NB recipients generated 13% CD41+ cells whereas cultures from BM recipients generated 22% CD41+ cells. However, by 4 weeks post-transplantation, the numbers of CD41+ cells were similar in cultures derived from NB recipients and BM recipients (Table 1). Moreover, at 2 and 4 weeks post-transplantation, there were fewer polyploid cells in liquid cultures from NB recipients compared to BM recipients (Table 1). The lower number of polyploid cells was commensurate with the lower number of CD41+ cells. This suggests that the rate of maturation of megakaryocyte is similar in NB and BM. In conclusion, our studies show that CB stem cells generate megakaryocytic progenitors at a slower rate than BM stem cells and that the delay in platelet recovery is not a result of a delay in the maturation of megakaryocytic progenitors. Thus, in order to increase the rate of platelet recovery following CB transplantation, the focus should be on enhancing the rate of production of megakaryocytic progenitors from hematopoietic stem cells. Table 1. Platelet(×103/μl) CFU-Meg(Colonies/1 × 105 Cells) CD41(%) Ploidy(% >4N DNAContent) normal BM(n=5) 1200±120 35.5±5 55±5 27±3 2 weeks NBT-2M(n=5) 190± 38 10±2 13±2 7.5±1 BMT-0.5M(n=5) 400±50 17±4 22±1 11±2 4 weeks NBT-2M(n=5) 550±59 18±2 26±2 14.5±2 BMT-0.5M(n=5) 730±110 23±3 27±1 18±3 8 weeks NBT-2M(n=5) 930±115 39±7 N/A N/A BMT-0.5M(n=5) 970±130 40±4 N/A N/A