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Evidence supporting the efficacy of in utero hematopoietic cell transplantation (IUHCT) in a valid large animal model is needed prior to clinical application. The objective of this study was to establish clinically relevant levels of hematopoietic chimerism in a canine model of maternal-to-fetal IUHCT. We first assessed immune and hematopoietic ontogeny relevant to IUHCT in the canine model and identified 40 days' gestation (term 63 days) as a time point at the initiation of thymic selection, and prior to bone marrow hematopoiesis, that might be optimal for IUHCT. We next determined that intravascular administration of donor cells via intracardiac injection was far more efficient and resulted in much higher levels of donor cell engraftment than intraperitoneal injection. By applying these findings, we achieved stable long-term multilineage engraftment in 21 of 24 surviving recipients with an average level of initial chimerism of 11.7% (range 3% to 39%) without conditioning or evidence of graft-versus-host disease. Donor cell chimerism remained stable for up to 2 years and was associated with donor-specific tolerance for renal transplantation. The levels of donor cell chimerism achieved in this study would be therapeutic for many hematopoietic disorders and are supportive of a clinical trial of IUHCT., (© 2014 by The American Society of Hematology.)

Although platelets appear by embryonic day 10.5 in the developing mouse, an embryonic role for these cells has not been identified. The SYK-SLP-76 signaling pathway is required in blood cells to regulate embryonic blood-lymphatic vascular separation, but the cell type and molecular mechanism underlying this regulatory pathway are not known. In the present study we demonstrate that platelets regulate lymphatic vascular development by directly interacting with lymphatic endothelial cells through C-type lectin-like receptor 2 (CLEC-2) receptors. PODOPLANIN (PDPN), a transmembrane protein expressed on the surface of lymphatic endothelial cells, is required in nonhematopoietic cells for blood-lymphatic separation. Genetic loss of the PDPN receptor CLEC-2 ablates PDPN binding by platelets and confers embryonic lymphatic vascular defects like those seen in animals lacking PDPN or SLP-76. Platelet factor 4-Cre-mediated deletion of Slp-76 is sufficient to confer lymphatic vascular defects, identifying platelets as the cell type in which SLP-76 signaling is required to regulate lymphatic vascular development. Consistent with these genetic findings, we observe SLP-76-dependent platelet aggregate formation on the surface of lymphatic endothelial cells in vivo and ex vivo. These studies identify a nonhemostatic pathway in which platelet CLEC-2 receptors bind lymphatic endothelial PDPN and activate SLP-76 signaling to regulate embryonic vascular development.