Facilitation of Thymic and Bone Marrow Engraftment by Hematopoietic Progenitors Expanded Ex-Vivo Using the Notch Ligand Delta1

Previously, we demonstrated that activation of Notch receptors by culture of murine lin⁻ Sca-1⁺c-kit⁺ (LSK) hematopoietic progenitors with the Notch ligand Delta1^ext-IgG, consisting of the extracellular domain of Delta1 fused to the Fc domain of human IgG, promoted early T cell differentiation and increased the number of progenitors capable of short-term lymphoid and myeloid reconstitution. Here we show accelerated thymic engraftment and T cell recovery after hematopoietic cell transplantation (HCT) in recipients of LSK cells cultured with Delta^1ext-IgG compared to recipients of non-cultured LSK cells or control human IgG-cultured LSK cells. Furthermore, data suggest that addition of Delta1^ext-IgG cultured LSK cells to non-cultured LSK cells have the potential to accelerated T cell recovery by the non-cultured LSK cells by facilitating thymic engraftment by the non-cultured LSK cells. 10³ Ly5a LSK cells cultured with Delta1^ext-IgG for 21 days generated a 10⁸-fold increase in total number of cells compared to culture with control IgG. Lethally irradiated C57Bl/6 mice received 10⁵ C57Bl/6 GFP⁺ LSK cells alone or along with 10³ Ly5a LSK cells or 10⁶ Lya5a Delta1^ext-IgG-cultured cells or 10⁶ Ly5a IgG cultured cells. We monitored egraftment by difference in CD45 as well as GFP. Mice were sacraficed and blood, bone marrow and thymus were harvested at 1,2,3 and 4 wks after HCT. At 3 wks after HCT, blood samples from recipients of Delta1^ext-IgG-cultured cells contained at least 4-fold higher total number of CD3⁺ cells compared to the other control groups (p<0.05). Furthermore, recipients of Delta1^ext-IgG-cultured cells contained at least 3-fold higher numbers of CD3⁺ cells derived from the GFP+ LSK cells compared to the other control groups (p<0.05). 1 wk after HCT, thymuses and BM from recipients of Delta1^ext-IgG-cultured cells showed at lest a 3- and 7-fold increase, respectively, in the number of GFP⁺ cells compared to the other control groups (p<0.05and p<0.007). Previously, we have shown our system generates committed and non-committed T cell progenitors with enhanced T cell reconstitution potential. Here, we demonstrate that the addition of Delta1^ext-IgG-cultured LSK cells to a non-cultured LSK graft has the potential to facilitate BM and thymic engraftment of the non-cultured LSK cells. By using the GFP⁺ LSK cells, we were able to demonstrate that the facilitation of early BM and thymic recovery was due to the increased engraftment/homing of the GFP⁺ cells rather than autologous recovery of the host. Thus this report describes a novel and clinically feasible ex-vivo culture system using Delta1 for the generation of T cell progenitors as a means to accelerate initial T-cell recovery as well as facilitating BM and thymic engraftment after HCT.