Timing of Mesenchymal Stem Cell Co-Transplantation and Site of Bone Engraftment Determine Levels of Hematopoietic Engraftment and Contribution of HSC to the Bone Niche

Abstract 940

We and others have previously demonstrated that the co-transplantation of marrow-derived stromal cells (MSC) with hematopoietic stem cells (HSC) results in higher levels of engraftment and acceleration of the rate of appearance of donor derived hematopoietic cells in the peripheral blood (PB) following transplantation. Possible explanations for this effect are MSC immunomodulatory properties and/or the ability of MSC to produce factors which support the transplanted HSC or prevent them from undergoing apoptosis. Here, we hypothesized that transplanted MSC are able to engraft within the recipient's bone marrow and integrate into vascular and/or osteoblastic niches to selectively create HSC donor optimized sites, and thereby enhance the rate and level of donor-derived hematopoietic reconstitution. Using an allogeneic sheep-to-sheep in-utero transplantation model, we administered intra-peritoneally 1.4×10^5 CD34+ cells transduced with a lentiviral vector encoding eGFP, (eGFP-CD34+) in combination with 2.5×10^5 same donor MSC transduced with a lentiviral vector encoding mKate (mKateMSC) (n=4). Another group of animals (n=4) received 2.5×10^5 mKateMSC, 3 days prior to transplantation of same donor 1.4×10^5 eGFP-CD34+. At 60 days post-transplant we performed flow cytometric analysis and Colony Forming Unit assay (CFU) of PB and BM to assess the levels of donor cell engraftment. Confocal microscopic analysis of bone sections was also performed in order to identify the localization and interaction between the transplanted HSC and MSC. Animals receiving mKateMSC 3 days prior to HSC transplantation displayed a 1.6 and a 1.1 fold increase in circulating donor GFP+cells and donor GFP+BM cells, respectively than animals receiving MSC+HSC simultaneously. However the latter had significantly higher levels of CD34 engraftment in BM (4.5-fold) than animals receiving mKateMSC 3 days prior to HSC transplantation. This also corresponded to higher levels of GFP+CFU in animals transplanted with MSC+HSC simultaneously. Confocal microscopy revealed that regardless of whether animals received mKateMSC 3 days prior to HSC or MSC+HSC simultaneously, HSC and MSC engrafted in clusters; however, there was no preferential interaction of the transplanted HSC with autologous MSC over the recipient's own cells. Nevertheless, animals receiving mKateMSC 3 days prior to HSC had higher levels of MSC in their BM than animals receiving MSC+HSC simultaneously. Furthermore, independent of the regimen of cells transplanted, depending on the site of bone engraftment, i.e., metaphysis or diaphysis, transplanted HSC localized preferentially in perivascular areas in the diaphysis, while in the metaphysic they appeared to contribute to the osteoblastic cell layer coating the ossifying bone. Also, HSC contributed to the osteoblastic layer more consistently and robustly than the transplanted MSC. These results show that the delivery of MSC prior to HSC results in higher levels of MSC engraftment in the bone marrow and higher levels of donor derived blood cells in circulation. However, the presence of MSC in the transplanted graft is necessary for optimal engraftment of CD34+ cells. Furthermore, CD34+ cells, and not MSC, migrated efficiently to the metaphysis where they were able to integrate into the developing bone and contribute to the osteoblastic layer.