Influence of a dual-injection regimen, plerixafor and CXCR4 on in utero hematopoietic stem cell transplantation and engraftment with use of the sheep model.

BACKGROUND AIMS: Inadequate engraftment of hematopoietic stem cells (HSCs) after in utero HSC transplantation (IUHSCT) remains a major obstacle for the prenatal correction of numerous hereditary disorders. HSCs express CXCR4 receptors that allow homing and engraftment in response to stromal-derived factor 1 (SDF-1) ligand present in the bone marrow stromal niche. Plerixafor, a mobilization drug, works through the interruption of the CXCR4-SDF-1 axis. METHODS: We used the fetal sheep large-animal model to test our hypotheses that (i) by administering plerixafor in utero before performing IUHSCT to release fetal HSCs and thus vacating recipient HSC niches, (ii) by using human mesenchymal stromal/stem cells (MSCs) to immunomodulate and humanize the fetal BM niches and (iii) by increasing the CXCR4(+) fraction of CD34(+) HSCs, we could improve engraftment. Human cord blood-derived CD34(+) cells and human bone marrow-derived MSCs were used for these studies. RESULTS: When MSCs were transplanted 1 week before CD34(+) cells with plerixafor treatment, we observed 2.80% donor hematopoietic engraftment. Combination of this regimen with additional CD34(+) cells at the time of MSC infusion increased engraftment levels to 8.77%. Next, increasing the fraction of CXCR4(+) cells in the CD34(+) population albeit transplanting at a late gestation age was not beneficial. Our results show engraftment of both lymphoid and myeloid lineages. CONCLUSIONS: Prior MSC and HSC cotransplantation followed by manipulation of the CXCR4-SDF-1 axis in IUHSCT provides an innovative conceptual approach for conferring competitive advantage to donor HSCs. Our novel approach could provide a clinically relevant approach for enhancing engraftment early in the fetus.

Temporal definition of haematopoietic stem cell niches in a large animal model of in utero stem cell transplantation.

The fetal sheep model has served as a biologically relevant and translational model to study in utero haematopoietic stem cell transplantation (IUHSCT), yet little is known about the ontogeny of the bone marrow (BM) niches in this model. Because the BMmicroenvironment plays a critical role in the outcome of haematopoietic engraftment, we have established the correlation between the fetal-sheep and fetal-human BM niche ontogeny, so that studies addressing the role of niche development at the time of IUHSCT could be accurately performed. Immunofluorescence confocal microscopic analysis of sheep fetal bone from gestational days (gd) 25-68 showed that the BM microenvironment commences development with formation of the vascular niche between 25 and 36 gd in sheep; correlating with the events at 10-11 gestational weeks (gw) in humans. Subsequently, between 45 and 51 gd in sheep (c. 14 gw in humans), the osteoblastic/endosteal niche started developing, the presence of CD34(+)  CD45(+) cells were promptly detected, and their number increased with gestational age. IUHSCT, performed in sheep at 45 and 65 gd, showed significant haematopoietic engraftment only at the later time point, indicating that a fully functional BM microenvironment improved engraftment. These studies show that sheep niche ontogeny closely parallels human, validating this model for investigating niche influence/manipulation in IUHSCT engraftment.

Generation of CD34+ cells from human embryonic stem cells using a clinically applicable methodology and engraftment in the fetal sheep model.

Until now, ex vivo generation of CD34(+) hematopoietic stem cells (HSCs) from human embryonic stem cells (hESCs) mostly involved use of feeder cells of nonhuman origin. Although they provided invaluable models to study hematopoiesis, in vivo engraftment of hESC-derived HSCs remains a challenging task. In this study, we used a novel coculture system composed of human bone marrow-derived mesenchymal stromal/stem cells (MSCs) and peripheral blood CD14(+) monocyte-derived macrophages to generate CD34(+) cells from hESCs in vitro. Human ESC-derived CD34(+) cells generated using this method expressed surface makers associated with adult human HSCs and upregulated hematopoietic stem cell genes comparable to human bone marrow-derived CD34(+) cells. Finally, transplantation of purified hESC-derived CD34(+) cells into the preimmune fetal sheep, primed with transplantation of MSCs derived from the same hESC line, demonstrated multilineage hematopoietic activity with graft presence up to 16 weeks after transplantation. This in vivo demonstration of engraftment and robust multilineage hematopoietic activity by hESC-derived CD34(+) cells lends credence to the translational value and potential clinical utility of this novel differentiation and transplantation protocol.