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.
No relevant conflicts of interest to declare.
During ontogeny, definitive hematopoietic stem/progenitor cells (HSC) are thought to arise from vascular endothelial cells, through an endothelial-to-hematopoietic transition, a natural process that ...occurs in unique, specialized embryonic hemogenic endothelial cells. Developmental studies, and experiments using pluripotent stem cells in an effort to recapitulate this process and thereby gain a better understanding of the emergence of definitive hematopoiesis, have collectively led to the prevailing view that the hemogenic endothelium constitutes a transient population of cells within the embryo that rapidly disappears during development and is absent in the adult. Herein, we provide the first evidence that at early time points of gestation, prior to the establishment of hematopoiesis, a unique subpopulation of Stro-1+ cells present within the inner part of the developing human bone marrow co-expresses APLNR, a marker of angiogenic mesoderm. Moreover, these Stro-1+APLNR+ cells express multiple other markers described for hemogenic endothelium, and subsequently contribute to the vasculature, cartilage, and bone. Importantly, we also show that cells expressing these same markers of primitive mesoderm/hemogenic endothelium persist at low frequency within the adult marrow. These adult-derived cells can be extensively expanded in vitro without loss of potential, but lack hematopoietic colony-forming potential in vitro. However, upon transplantation into a fetal microenvironment, clonally-derived populations of these adult Stro1+ isolated stromal progenitors (SIPs) not only contribute to the vasculature and nascent BM niches, but also efficiently generate, at a clonal level, hematopoietic stem cells (HSC) that are capable of robust, multilineage hematopoietic reconstitution, with generation of both myeloid and lymphoid cells upon serial transplantation. In conclusion, our studies have thus uncovered the latent potential of a highly expandable population of seemingly vestigial adult human somatic cells, whose ontogenic history includes a phenotype identical to that described for hemogenic endothelium. We have also shown that, if provided with the appropriate/necessary inductive factors, these unique adult cells are capable of giving rise to hematopoietic cells that fulfill the gold standard criteria for bona fide HSC. Therefore, these cells could potentially be more amenable to reprogramming technologies, to produce HSC that could be used to treat/cure a broad variety of blood diseases.
No relevant conflicts of interest to declare.
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