Optimization Of Vascular Niches To Increase Hematopoietic Engraftment

In utero hematopoietic stem cell transplantation (IUHSCT) is a promising approach for correcting selected congenital hematologic and immunologic disorders. However, if higher levels of donor hematopoietic stem/progenitor (HSC) cell engraftment could be achieved, a wider range of inherited disorders could be targeted. We have previously shown that adult bone marrow (BM) derived-CD34+ cells adhere less efficiently to fetal stromal cells than to their adult counterpart. Furthermore, it has been shown that perivascular cells are able to support, through cellular interactions, the long-term engrafting HSC. Here, we hypothesized that by transplanting bone marrow (BM)-derived endothelial progenitor cells (EPC) prior to HSC transplantation, it would be possible to establish HSC donor-optimized vascular niches within the recipient’s BM, and thereby enhance the rate and level of donor-derived hematopoietic reconstitution. Adult sheep BM HSC were immunoselected with an antibody against sheep CD34, while EPC were isolated by adherence to collagen type I. Characterization of these cells demonstrated that they were spindle-shaped, and they expressed fetal liver kinase (flk-1/KDR), vonWillebrand factor (vWF), and melanoma cell adhesion molecule (MCAM/CD146). In addition, these cells formed capillary-like structures in Matrigel-based media. Using an allogeneic sheep-to-sheep in-utero transplantation model, we administered, intraperitoneally, 1.4X10⁵ CD34⁺ cells transduced with an eGFP-encoding lentiviral vector (HSCeGFP) in combination with 7.1X10⁵ EPC transduced with an mKate-encoding lentiviral vector (EPCmKate) (n=4), from the same donor, either concurrently, or 3 days prior to HSCeGFP transplantation. At 60 days post-transplant, we performed flow cytometry on peripheral blood (PB) and BM to assess the levels of donor cell engraftment. We also performed confocal microscopic analysis of bone sections to identify the localization and interaction between transplanted cells. Our results demonstrate that animals receiving EPCmKate 3 days prior to HSC transplantation displayed 13-fold higher levels of eGFP(+) hematopoietic cells in their BM (6.5±0.5%), when compared with animals receiving EPC and HSC simultaneously (0.39±0.29%). Confocal microscopy analysis showed that, regardless of the time-point of transplant, donor cells that engrafted in the diaphysis localized to the perivascular area, and a correlation was found between the levels of CD146(+)mKate cells and HSCeGFP engraftment. By contrast, in the metaphysis, only eGFP(+) cells were detected, and these cells co-expressed osteopontin, a marker of osteoblasts. These results show that in IUHSCT, delivery of EPC,CD146(+), cells prior to CD34+HSC results in modification of the vascular niches by donor-derived cells, leading to significantly higher levels of HSC engraftment. Furthermore, a considerable percentage of CD34+eGFP(+) cells did not contribute to the hematopoietic pool, but rather, contributed to the developing bone, suggesting that a more effective selective process for HSC might be necessary for improving engraftment in IUHSCT.