• In utero injection of an antibody against the c-Kit receptor can effectively deplete host HSCs in mice.• In utero depletion of host HSCs leads to significantly increased engraftment after neonatal congenic hematopoietic cell transplantation.Although in utero hematopoietic cell transplantation is a promising strategy to treat congenital hematopoietic disorders, levels of engraftment have not been therapeutic for diseases in which donor cells have no survival advantage. We used an antibody against the murine c-Kit receptor (ACK2) to deplete fetal host hematopoietic stem cells (HSCs) and increase space within the hematopoietic niche for donor cell engraftment. Fetal mice were injected with ACK2 on embryonic days 13.5 to 14.5 and surviving pups were transplanted with congenic hematopoietic cells on day of life 1. Low-dose ACK2 treatment effectively depleted HSCs within the bone marrow with minimal toxicity and the antibody was cleared from the serum before the neonatal transplantation. Chimerism levels were significantly higher in treated pups than in controls; both myeloid and lymphoid cell chimerism increased because of higher engraftment of HSCs in the bone marrow. To test the strategy of repeated HSC depletion and transplantation, some mice were treated with ACK2 postnatally, but the increase in engraftment was lower than that seen with prenatal treatment. We demonstrate a successful fetal conditioning strategy associated with minimal toxicity. Such strategies could be used to achieve clinically relevant levels of engraftment to treat congenital stem cell disorders. (Blood. 2014;124(6):973-980) IntroductionHematopoietic stem cell (HSC) transplantation is a promising strategy to treat many nonmalignant genetic disorders such as hemoglobinopathies, immunodeficiencies, and inborn errors of metabolism, 1 and may even provide tolerance for solid organ transplants.2 However, this approach often requires host myeloablation and immunosuppression, which carries significant morbidity. 3,4 Transplantation into the immunologically naive fetal environment to circumvent the host immune response is an attractive alternative strategy to achieve sustained engraftment and donor-specific tolerance. This approach of in utero hematopoietic cell transplantation (IUHCTx) has been successful in many animal models, but clinical applications remain hampered by low levels of engraftment that are not sufficient to ameliorate symptoms or cure most diseases (reviewed in Nijagal et al 5 ). Because the only clinical successes have been achieved in fetuses with severe combined immunodeficiency, [6][7][8] it has been suggested that success is limited by barriers such as rejection of the stem cell graft and lack of space within the hematopoietic niche (reviewed in Flake and Zanjani 9 ). We and others have previously explored the role of an immune response to donor cells and reported that the maternal immune system is a significant barrier to engraftment. 10,11 The fetal host can become tolerant to transplanted cells through clonal de...
1326 Using hESC differentiation system we recently identified a mesenchymoangioblast (MAB) as a novel precursor for mesenchymal stem cells (MSCs) and endothelial cells and demonstrated that mesenchymal and hematopoietic cells develop sequentially from mesodermal precursors with primary angiogenic potential – MAB and hemangioblasts (HB), respectively. In addition, we found that angiogenic mesoderm reminiscent of lateral plate/extraembryonic mesoderm in the embryo can be identified by surface expression of apelin receptor (APLNR) and lack of expression of typical endothelial (CD31, CD144), hematopoietic (CD43, CD45) and mesenchymal (CD73, CD195) markers i.e. as EHMlin-APLNR+ cells. (Vodyanik et al. Cell Stem Cell 2010;7:718). In response to FGF2 APLNR+ cells form morphologically distinct compact mesenchymal or MAB colonies and disperse hematopoietic or blast (HB) colonies in serum-free clonogenic semisolid medium. When transferred to the adherent cultures in serum-free medium with FGF2, individual colonies gave rise to multipotential mesenchymal cell lines with typical phenotype (CD146+CD105+CD73+CD31-CD43-45-), differentiation (chondro-, osteo-, and adipogenesis) and robust proliferation (>80 doublings) potentials. In contrast, HB colonies consisted almost entirely of CD235a and CD41a expressing cells with morphology resembling erythroblasts. Replating of HB colonies in hematopoietic serum-free and serum-containing clonogenic medium demonstrated that they gave rise to erythroid, megakaryocytic and mixed colonies composed of erythroid, megakaryocytic cells and macrophages indicating that BL-CFCs probably reflected the first wave of embryonic hematopoiesis initiated in the yolk sac. To define pathways involved in MAB and HB development we tested the effect of different growth factors and cytokines on mesenchymal and blast colony formation. We confirmed that the development of MAB and HB depends on FGF2, by demonstrating complete suppression of blast and mesenchymal colony formation by elimination of FGF2 from clonogenic culture or by abrogation of FGF2 signaling using PD 173074 inhibitor of FGF2 receptor autophosphorylation. The formation of MAB and HB colonies was also completely abrogated by adding TGFb or activin A, and increased in the presence of SB431542 TGFb signaling inhibitor. PDGF-BB alone lacked colony-forming activity, but its addition to FGF2 significantly increased the frequency and size of mesenchymal but not blast colonies. In contrast, the addition of VEGF essentially abrogated mesenchymal colony formation. Although VEGF had little effect on BL-CFCs, the addition KI8751 KDR inhibitor significantly decreased the number of blast colonies, confirming that their development depends on VEGF signaling. The addition of individual hematopoietic cytokines to FGF2 had a relatively mild effect on the number of blast colonies. However, they increased the size of colonies, which was especially obvious with the addition of EPO. When IL3, IL6, EPO, TPO, and SCF were added to clonogenic cultures together with FGF2, we observed a significant increase in the number and size of blast colonies, some of which had grown into very large grape-like structures. Because we found that emerging CD144+CD235a+ cells generated hematopoietic colonies morphologically resembling blast colonies in the presence of FGF2 and hematopoietic cytokines, we concluded that hematopoietic cytokine-free clonogenic cultures would be more appropriate for detection of BL-CFCs and enhancing the specificity of this assay. The finding that mesenchymal and blast CFCs arise from cells expressing APLNR prompted us to test whether this receptor is involved in the regulation of MAB and HB development. We found that APLNR agonist aplein-12 inhibits mesenchymal colonies, while it significantly increases the formation of blast colonies in synergy with VEGF. Together, these studies demonstrated that the activation of multiple but different signaling pathways regulates the formation of mesenchymal and blast colonies. Disclosures: Slukvin: CDI: Consultancy, Equity Ownership.
Introduction In utero hematopoietic cell transplantation (IUHCTx) is a promising strategy to treat congenital disorders as the fetal host can potentially be tolerized to transplanted cells early in gestation. However, levels of engraftment have been low and fetal host conditioning strategies to increase space in hematopoietic niches have not been widely explored. We hypothesized that depletion of fetal host hematopoietic stem cells (HSC) using an antibody against the c-kit receptor (ACK2), a strategy which selectively depletes HSC by disrupting stem cell factor (SCF) signaling, would improve engraftment after HSC transplantation. Methods Fetal C57B6.CD45.2 (B6) mice were injected with increasing doses of ACK2 (2.5-50 µg/fetus) or isotype control antibody on E14.5 and surviving pups were transplanted with congenic B6.CD45.1 fetal liver mononuclear cells (2.5×106 cells/pup) on day of life 1 (P1, 7 days after in utero injection), allowing post-transplantation host monitoring. Host HSC depletion and residual serum ACK2 concentration were examined on P1. Peripheral blood chimerism, defined as donor/(donor+host) CD45 cells, as well as the lineage distribution of chimeric cells, were determined beginning 4 weeks after transplantation. Results Survival to birth among fetuses injected with 2.5, 5, or 10 µg of ACK2 was similar to controls (control: 74%; 2.5 µg: 80%; 5 µg: 71%; 10 µg: 60%, p=0.2 by chi-square test, n≥45/group) but was significantly lower at higher concentrations (20 µg: 37%; 50 µg: 31%, p<0.001 vs. control, n≥70/group). Transient anemia and leukopenia were observed on P1 with doses ≥ 5 µg which resolved by P7 (n=17). Four of 19 pups previously treated with ACK2 (2.5-10 µg) and observed long-term had patchy coat discoloration, possibly a manifestation of disruption of C-kit+ melanocyte migration. In utero ACK2 treatment resulted in significant and dose-dependent depletion of host HSCs (defined as Lin-Sca-1+C-kit+, KLS) in the bone marrow of treated animals by P1 (Figure 1A). There was no depletion of KLS cells in the liver. Residual ACK2 antibody was undetectable in the serum by P1, validating our strategy of in utero depletion and neonatal transplantation. In animals receiving neonatal transplantation, ACK2 depletion resulted in a significant increase in levels of engraftment 4 weeks after transplantation compared to controls (control: 3.3±0.3%; 2.5 µg: 13±1.4%; 5 µg: 10±2.4%; 10 µg: 11±2.0%, p<0.05 for each dose vs control by ANOVA). Accordingly, we detected an increased number total bone marrow KLS cells 7 days after transplantation in ACK2 treated animals compared to controls (412±45.9 vs. 933±112 cells, p=0.01, n≥3/group). Moreover, levels of chimerism increased over time in treated animals (Figure 1B; 12 weeks: 2.5 µg: 190%; 5 µg: 170%; 10 µg: 160%) while they remained unchanged in controls. Overall, levels of chimerism achieved with ACK2 treatment were significantly higher than that observed in animals that received in utero transplantation without ACK2 depletion. Lineage analysis of peripheral blood for granulocytes, B cells, and T cells indicated an equal increase in all lineages, suggesting ACK2 depletes true HSCs and not committed progenitors. Interestingly, ACK2 depletion at doses 2.5-10 µg did not result in engraftment of allogeneic BALB/c cells (n=11), indicating that allogeneic neonatal transplantation, unlike in utero transplantation, is limited by a host immune response which is unaffected by ACK2. Conclusion We have demonstrated that fetal HSC depletion using ACK2 can lead to clinically relevant levels of donor cell engraftment with minimal toxicity. In previous studies with this antibody, host HSC depletion required either immunodeficient animals or concurrent irradiation, whereas we achieved depletion in wild-type fetal hosts, suggesting differences in fetal vs. adult HSC sensitivity to SCF signaling. Future studies should explore this strategy to improve engraftment in large animals models of IUHCTx. Disclosures: Weissman: Amgen, Systemix, Stem cells Inc, Cellerant: Consultancy, Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.
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