Potential Long-Term Treatment of Hemophilia A by Neonatal Co-Transplantation of Cord Blood Derived Endothelial Colony-Forming Cells and Placental Mesenchymal Stromal Cells

Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for cellbased gene therapy, but successful treatment has been hampered by insufficient engraftment of potential therapeutic cells. In this study, we sought to determine whether co-transplantation of endothelial colonyforming cells (ECFCs) and placenta-derived mesenchymal stromal cells (PMSCs) can achieve long-term engraftment and FVIII expression. ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector and luciferase, green fluorescent protein or Td-Tomato containing lentiviral tracking vectors. They were transplanted intramuscularly into neonatal or adult immunodeficient mice. In vivo bioluminescence imaging showed that the ECFC only and the cotransplantation groups but not the PMSCs only group achieved long-term engraftment for at least 26 weeks, and the co-transplantation group showed a higher engraftment than the ECFC only group at 16 and 20 weeks of age. In addition, cell transplantation at the neonatal age achieved higher engraftment than at the adult age. Immunohistochemical analyses further showed that the engrafted ECFCs expressed FVIII, maintained endothelial phenotype and generated functional vasculature. Next, cotransplantation of ECFCs and PMSCs into F8 knock-out HA mice reduced the blood loss volume from 562.13±19.84µl to 155.78±44.93µl in a tail-clip assay. This work demonstrated that co-transplantation of ECFCs with PMSCs at the neonatal age is a potential strategy to achieve stable, long-term engraftment, and thus holds great promise for cell-based treatment of HA. Funding Statement: This work was in part supported by UC Davis Department of Surgery departmental fund, and the UC Davis Medical Center Interdepartmental Seed Grant. Declaration of Interests: The authors declare: "None." Ethics Approval Statement: All animal procedures were approved by The University of California, Davis (UCD) institutional animal care and use committee (IACUC). All facilities used during the study period were accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International (AAALAC).