An Endothelial Cell-targeted D1R Fusion Protein Effectively Promotes Hematopoietic Stem And Progenitor Cell Engraftment In Both Bone Marrow And Liver

Recently, hematopoietic stem cell transplantation（HSCT）has become an importanttherapy for patients with hematonosis and hematologic malignancy.Umbilical cord blood(UCB), marrow and mobilized peripheral blood have been used successfully as principlesources of HSCT.UCB has become more attractive for its abundance, ready availabilityand immature immunogenicity compared to marrow or mobilized peripheral blood.Butumbilical cord blood transplantation (UCBT) in adults and high weigh patients is prevented by a limited number of hematopoietic stem cells (HSC) in each graft, whichleads to delayed engraftment and increased rates of infectious complications.Strategies toget sufficient HSCs for transplantation have been an important problem demandingprompt solution.A growing body of work has identified that the Notch pathway plays a fundamentalrole in regulating stem cell fate decisions to self-renew or differentiate.Notch receptorsand ligands are widely expressed in hemopoietic system.Notch activation is able tomaintain self-renewal of HSCs,which will be a novel thought to gain sufficient HSCs.Wehave developed an ex vivo expansion system for hematopoietic stem and progenitor cells(HSPCs) that used human umbilical vein endothelial cells (HUVECs) as feeder cells tomimic hematopoietic microenvironment supplemented with cytokines(SCF,FL,TPO,IL-6,IL-3) and endothelial cells-targeted Notch ligand （D1R,humanD1R）.Importantly, through in vivo D1R administration we assessed its influence onHSPCs engraftment in the bone marrow, the liver and hematopoietic microenvironment,and further analyzled the related mechanisms of Notch activation by theendothelium-targeted D1R in keeping self-renewal of HSCs and promoting engraftment.The main finds are as follows:1. The Notch-mediated ex vivo expansion system has been established and resulted in amarked increase in mouse HSPCs capable of enhanced long-term repopulation in themarrow.These suggested that Notch activation by D1R maintain the “stemness” of HSCsand promote expansion.2. Systemic administration of D1R to irradiated mice protected marrowmicroenvironment from the irradiation and caused a pronounced expansion of remnantbone marrow stem and progenitor cells in vivo, which were able to repopulate the marrow.3. D1R administration effectively promoted HSCs engraftment in both bone marrowand liver of mice receiving BM transplantation.Futher study found the formation ofvascular niche-like structures in the liver of recipient mice, which resulted in donor HSPCsexpansion, improved mouse survival and decreased the lowest effective transplant cellsdose keeping hematopoietic reconstitution. 4. Treatment of human cord blood CD34+cells with hD1R also substantially increasedcell counts in the culture system,which were capable of enhanced repopulation in themarrow of immunodeficient nonobese diabetic–severe combined immunodeficient(NOD-SCID) mice.In conclusion, the Notch-mediated ex vivo expansion system has successfully beenestablished and markedly increased HSPCs.We first used endothelial cells-targeted Notchligand D1R in vivo and observed its influence on HSPCs engraftment and hematopoieticmicroenvironment. Furthermore, we confirmed the role of Notch activation by using anendothelium-targeted Notch ligand in maintaining self-renewal of HSCs and promotingengraftment.