Abstract
INTRODUCTION: Use of HLA-mismatched umbilical cord blood (UCB) allows transplant of patients who lack related HLA identical or matched unrelated donors. A major disadvantage of UCB is the limited number of stem cells available within the graft, a contributor to delayed engraftment, especially in adults. Two strategies have been adopted clinically in an attempt to overcome this barrier. 1. ex vivo expansion of some or all of critical graft subfractions; 2. transplantation of two different UCB grafts. These two strategies are not mutually exclusive and are also sometimes used in combination. Little is known about the basic biology that underlies the differential migration and homing of the graft components. To study the biology of complex UCB transplants, we have developed a small animal in vivo imaging model.
METHODS: CD34-selected UCB cells were transduced overnight with a lentiviral construct consisting of GFP or a firefly luciferase/GFP fusion. After 24 hours, SCF, FLT3, G-CSF, and thrombopoietin were added and the cells were cultured for 3 to 8 days. All cultured cells were then injected into non-lethally irradiated, immunocompetent mice (NOD/SCID/IL-2Rγ − /−). Transduced cells were tracked weekly by bioluminescent imaging.
RESULTS: On culture day 3, >99% of cells remained CD34+ and no expansion was observed. After expansion in culture between days 3 and 8, 19% +/− 18.5% of transduced cells were GFP+CD45+ (n= 8). Cells cultured for three days (non-expanded) (3-11x105 CD34+ cells/3-8.6x104 GFP+) were able to create a detectable engraftment signal by post-transplant day 8. The engraftment signal was delayed until day 10 when an 8-fold excess of the expanded cultured cells (4.2x106 total consisting of 1.21x106 CD34+ cells/2.3x105 GFP+ and 6.51x105 CD34-GFP+ cells) were injected. Long term engraftment (>30 days) was unaffected by expansion in culture. However, expanded UCB exhibited a differential pattern of homing and engraftment in comparison to unexpanded UCB. Mice receiving unexpanded UCB exhibited a strong engraftment signal from the area of the calvarium in addition to signals from other marrow spaces. The calvarium signal was only weakly and intermittently observed among mice who received the expanded UCB graft. Pathologic evaluation of these areas is in progress.
CONCLUSIONS: 1. Expanded cells were capable of supporting engraftment, though at a slower rate than their unexpanded counterparts. This delay might ultimately be addressed through the optimization of the expansion technique. 2. The physiologic significance of the differential engraftment pattern(s) is unclear and will require further study.
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