Defining Engraftment Patterns and Interactions Between Hematopoietic Stem Cells and Stromal Cells after in Utero Transplantation

In utero hematopoietic stem cell transplantation (IUHSCT) has only been fully successful in recipients with severe combined immunodeficiency disorders. Because IUHSCT must be performed without myeloablation or immunosuppression, immunologic barriers and absence of stress-induced signaling have been considered significant contributors to the limited success of this procedure. Other potential barriers to successful engraftment of allogeneic adult donor hematopoietic stem/progenitor cells (HSC) in a fetal recipient result from the unique characteristics of fetal HSC biology and the fetal microenvironment. Transplanted adult cells could be out-competed by fetal HSC, and the fetal microenvironment might not support engraftment and/or expansion of HSC derived from ontogenically disparate sources, as differences in membrane composition and response to cytokines exist between fetal and adult cells.  We have previously shown that increased levels of HSC engraftment can be achieved in a fetal setting, if bone marrow (BM)-derived mesenchymal stromal cells (MSC) or (BM)-derived endothelial progenitor cells (EPC) are administered prior to, or at the time of, HSC transplantation. Using a fetal sheep model of IUHSCT, we interrogated mechanisms by which transplanting sheep EPC or MSC prior to, or concurrently with, sheep HSC leads to statistically significant higher levels of HSC engraftment when compared to HSC alone. Using confocal microscopy and fluorescently-labeled cells, we first determined the sites of engraftment/localization of donor EPC, MSC, and HSC at 2-2.5 months after IUHSCT when transplanted at the does of 7.1 x 10⁶/Kg, 2.5 x 10⁶/kg, and 2.1 x 10⁶ /kg, respectively, at 60-65 days of gestation (human equivalent of 16 gestational weeks). We demonstrated that EPC and MSC engraft at different sites after IUHSCT than the transplanted HSC. MSC and EPC engrafted in the diaphysis, where they integrated into the vascular and perivascular niches and produced SCF and CXCL12. Confocal analysis demonstrated that transplanted EPC produced higher levels of CXCL12 than endogenous cells, explaining, at least in part, the higher levels of hematopoietic engraftment seen in the BM and PB of animals transplanted with EPC 3 days prior to HSC. HSC engrafted mainly in the metaphysis, a metabolically active tissue in the fetus, which is comprised of large sinusoidal veins expressing high levels of hyaluronan (HA) synthase. In addition, donor HSC in the metaphysis were actively cycling (>90%) as determined by Ki67 staining. Fewer donor HSC were found in the diaphysis, and only a small percentage of these cells were dividing, but they localized in clusters in close proximity to donor MSC or EPC. Moreover, a direct correlation was found between the levels of donor-derived perivascular cells engrafted within the vasculature, and the numbers of donor-derived HSC present in the perivascular areas (defined as being within a distance of 5 cell nuclei from vessel), suggesting that, in the diaphysis, adult MSC and EPC provided support/attracted adult HSC. In summary, we have shown that, in a non-myeloablative fetal setting without stress-induced signaling, allogeneic adult donor HSC engraft and proliferate efficiently along with endogenous hematopoietic cells within the metaphysis, and that the high levels of CXCL12 produced by adult EPC were potentially responsible for their ability to significantly enhance HSC engraftment after IUHSCT.