Busulfan conditioning enhances engraftment of hematopoietic donor-derived cells in the brain compared with irradiation.
ABSTRACT: Hematopoietic stem cell gene therapy for neurological disorders relies on transmigration of donor-derived monocytes to the brain, where they can engraft as microglia and deliver therapeutic proteins. Many mouse studies use whole-body irradiation to investigate brain transmigration pathways, but chemotherapy is generally used clinically. The current evidence for transmigration to the brain after chemotherapy is conflicting. We compared hematopoietic donor cell brain engraftment after bone marrow (BM) transplants in busulfan- or irradiation-conditioned mice. Significantly more donor-derived microglial cells engrafted posttransplant in busulfan-conditioned brain compared with the irradiated, in both the short and long term. Although total Iba-1(+) microglial content was increased in irradiated brain in the short term, it was similar between groups over long-term engraftment. MCP-1, a key regulator of monocyte transmigration, showed long-term elevation in busulfan-conditioned brain, whereas irradiated brains showed long-term elevation of the proinflammatory chemokine interleukin 1? (IL-1?), with increased in situ proliferation of resident microglia, and significant increases in the relative number of amoeboid activated microglia in the brain. This has implications for the choice of conditioning regimen to promote hematopoietic cell brain engraftment and the relevance of irradiation in mouse models of transplantation.
Project description:Insufficient expression of factor VIII (fVIII) is a major hurdle in the development of successful nucleic acid treatments for hemophilia. However, we recently showed that under myeloablative and reduced-intensity total body irradiation (TBI) conditioning, transplantation of hematopoietic stem cells (HSCs) transduced with recombinant retroviruses containing B domain-deleted porcine fVIII (BDDpfVIII) sequences provides curative fVIII levels in a hemophilia A mouse model. In the current study, we tested BDDpfVIII activity after nonmyeloablative conditioning with busulfan, cyclophosphamide, or fludarabine and immunosuppressive agents CTLA4-Ig + anti-CD40L or anti-(murine)thymocyte serum (ATS). ATS is similar in action to anti-(human)thymocyte globulin (ATG), which is used clinically with busulfan in bone marrow transplantations to increase donor cell engraftment. Mice conditioned with busulfan + ATS and that received a transplant of BDDpfVIII-transduced stem-cell antigen 1-positive cells exhibited moderate levels of donor cell chimerism (between 20% and 60%) and achieved sustained fVIII levels more than 1 U/mL. Similar results were observed in mice preimmunized with human fVIII and conditioned with 5 Gy TBI + ATS or busulfan + ATS. These data demonstrate that it is possible to achieve sufficient fVIII expression after transplantation of BDDpfVIII-transduced HSCs following low-toxicity pretransplantation conditioning with targeted immunosuppression, potentially even in the context of preexisting inhibitors.
Project description:Although exceptionally high radiation dose-rates are currently attaining clinical feasibility, there have been relatively few studies reporting the biological consequences of these dose-rates in hematopoietic cell transplant (HCT). In zebrafish models of HCT, preconditioning before transplant is typically achieved through radiation alone. We report the comparison of outcomes in adult zebrafish irradiated with 20 Gy at either 25 or 800 cGy/min in the context of experimental HCT. In non-transplanted irradiated fish we observed no substantial differences between dose-rate groups as assessed by fish mortality, cell death in the kidney, endogenous hematopoietic reconstitution, or gene expression levels of p53 and ddb2 (damage-specific DNA binding protein 2) in the kidney. However, following HCT, recipients conditioned with the higher dose rate showed significantly improved donor-derived engraftment at 9 days post transplant (p ? 0.0001), and improved engraftment persisted at 31 days post transplant. Analysis for sdf-1a expression, as well as transplant of hematopoietic cells from cxcr4b -/- zebrafish, (odysseus), cumulatively suggest that the sdf-1a/cxcr4b axis is not required of donor-derived cells for the observed dose-rate effect on engraftment. Overall, the adult zebrafish model of HCT indicates that exceptionally high radiation dose-rates can impact HCT outcome, and offers a new system for radiobiological and mechanistic interrogation of this phenomenon. Key words: Radiation dose rate, Total Marrow Irradiation (TMI), Total body irradiation (TBI), SDF-1, Zebrafish, hematopoietic cell transplant.
Project description:Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beM?s) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beM?s to populate the niche and that the presence of beM?s does not alter behavior. Furthermore, beM?s maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beM?s as a unique CNS cell type and demonstrates that therapeutic engraftment of beM?s may be possible with irradiation-free conditioning regimens.
Project description:Recombinant human thrombopoietin (rHuTPO) is a drug that is used clinically to promote megakaryocyte and platelet generation. Here, we report the mitigative effect of rHuTPO (administered after exposure) against severe whole body irradiation in mice. Injection of rHuTPO for 14 consecutive days following exposure significantly improved the survival rate of lethally irradiated mice. RHuTPO treatment notably increased bone marrow cell density and LSK cell numbers in the mice after sub-lethal irradiation primarily by promoting residual HSC proliferation. In lethally irradiated mice with hematopoietic cell transplantation, rHuTPO treatment increased the survival rate and enhanced hematopoietic cell engraftment compared with the placebo treatment. Our observations indicate that recombinant human TPO might have a therapeutic role in promoting hematopoietic reconstitution and HSC engraftment.
Project description:BACKGROUND:Microglia, the primary resident myeloid cells of the brain, play critical roles in immune defense by maintaining tissue homeostasis and responding to injury or disease. However, microglial activation and dysfunction has been implicated in a number of central nervous system (CNS) disorders, thus developing tools to manipulate and replace these myeloid cells in the CNS is of therapeutic interest. METHODS:Using whole body irradiation, bone marrow transplant, and colony-stimulating factor 1 receptor inhibition, we achieve long-term and brain-wide (~?80%) engraftment and colonization of peripheral bone marrow-derived myeloid cells (i.e., monocytes) in the brain parenchyma and evaluated the long-term effects of their colonization in the CNS. RESULTS:Here, we identify a monocyte signature that includes an upregulation in Ccr1, Ms4a6b, Ms4a6c, Ms4a7, Apobec1, Lyz2, Mrc1, Tmem221, Tlr8, Lilrb4a, Msr1, Nnt, and Wdfy1 and a downregulation of Siglech, Slc2a5, and Ccl21a/b. We demonstrate that irradiation and long-term (~?6 months) engraftment of the CNS by monocytes induces brain region-dependent alterations in transcription profiles, astrocytes, neuronal structures, including synaptic components, and cognition. Although our results show that microglial replacement with peripherally derived myeloid cells is feasible and that irradiation-induced changes can be reversed by the replacement of microglia with monocytes in the hippocampus, we also observe that brain-wide engraftment of peripheral myeloid cells (relying on irradiation) can result in cognitive and synaptic deficits. CONCLUSIONS:These findings provide insight into better understanding the role and complexity of myeloid cells in the brain, including their regulation of other CNS cells and functional outcomes.
Project description:BACKGROUND:Chimeric mouse models generated via adoptive bone marrow transfer are the foundation for immune cell tracking in neuroinflammation. Chimeras that exhibit low chimerism levels, blood-brain barrier disruption and pro-inflammatory effects prior to the progression of the pathological phenotype, make it difficult to distinguish the role of immune cells in neuroinflammatory conditions. Head-shielded irradiation overcomes many of the issues described and replaces the recipient bone marrow system with donor haematopoietic cells expressing a reporter gene or different pan-leukocyte antigen, whilst leaving the blood-brain barrier intact. However, our previous work with full body irradiation suggests that this may generate a pro-inflammatory peripheral environment which could impact on the brain's immune microenvironment. Our aim was to compare non-myeloablative busulfan conditioning against head-shielded irradiation bone marrow chimeras prior to implantation of glioblastoma, a malignant brain tumour with a pro-inflammatory phenotype. METHODS:Recipient wild-type/CD45.1 mice received non-myeloablative busulfan conditioning (25?mg/kg), full intensity head-shielded irradiation, full intensity busulfan conditioning (125?mg/kg) prior to transplant with whole bone marrow from CD45.2 donors and were compared against untransplanted controls. Half the mice from each group were orthotopically implanted with syngeneic GL-261 glioblastoma cells. We assessed peripheral blood, bone marrow and spleen chimerism, multi-organ pro-inflammatory cytokine profiles at 12?weeks and brain chimerism and immune cell infiltration by whole brain flow cytometry before and after implantation of glioblastoma at 12 and 14 weeks respectively. RESULTS:Both non-myeloablative conditioning and head-shielded irradiation achieve equivalent blood and spleen chimerism of approximately 80%, although bone marrow engraftment is higher in the head-shielded irradiation group and highest in the fully conditioned group. Head-shielded irradiation stimulated pro-inflammatory cytokines in the blood and spleen but not in the brain, suggesting a systemic response to irradiation, whilst non-myeloablative conditioning showed no cytokine elevation. Non-myeloablative conditioning achieved higher donor chimerism in the brain after glioblastoma implantation than head-shielded irradiation with an altered immune cell profile. CONCLUSION:Our data suggest that non-myeloablative conditioning generates a more homeostatic peripheral inflammatory environment than head-shielded irradiation to allow a more consistent evaluation of immune cells in glioblastoma and can be used to investigate the roles of peripheral immune cells and bone marrow-derived subsets in other neurological diseases.
Project description:Whole brain irradiation remains important in the management of brain tumors. Although necessary for improving survival outcomes, cranial irradiation also results in cognitive decline in long-term survivors. A chronic inflammatory state characterized by microglial activation has been implicated in radiation-induced brain injury. We here provide the first comprehensive transcriptional profile of irradiated microglia. Fluorescence-activated cell sorting was used to isolate CD11b+ microglia from the hippocampi of C57BL/6 and Balb/c mice 1 month after 10 Gy cranial irradiation. Affymetrix gene expression profiles were evaluated using linear modeling and rank product analyses. One month after irradiation, a conserved irradiation signature across strains was identified, comprising 448 and 85 differentially up- and downregulated genes, respectively. Gene set enrichment analysis demonstrated enrichment for inflammation, including M1 macrophage-associated genes, but also an unexpected enrichment for extracellular matrix and blood coagulation-related gene sets, in contrast previously described microglial states. Weighted gene coexpression network analysis confirmed these findings and further revealed alterations in mitochondrial function. The RNA-seq transcriptome of microglia 24-h postradiation proved similar to the 1-month transcriptome, but additionally featured alterations in apoptotic and lysosomal gene expression. Reanalysis of published aging mouse microglia transcriptome data demonstrated striking similarity to the 1-month irradiated microglia transcriptome, suggesting that shared mechanisms may underlie aging and chronic irradiation-induced cognitive decline. GLIA 2015;63:754-767.
Project description:Peripherally derived macrophages infiltrate the brain after bone marrow transplantation and during central nervous system (CNS) inflammation. It was initially suggested that these engrafting cells were newly derived microglia and that irradiation was essential for engraftment to occur. However, it remains unclear whether brain-engrafting macrophages (beMφs) acquire a unique phenotype in the brain, whether long-term engraftment may occur without irradiation, and whether brain function is affected by the engrafted cells. In this study, we demonstrate that chronic, partial microglia depletion is sufficient for beMφs to populate the niche and that the presence of beMφs does not alter behavior. Furthermore, beMφs maintain a unique functional and transcriptional identity as compared with microglia. Overall, this study establishes beMφs as a unique CNS cell type and demonstrates that therapeutic engraftment of beMφs may be possible with irradiation-free conditioning regimens. Overall design: Mice were given 1000rad whole body irradiation, followed by bone marrow transplant with UBC-GFP bone marrow at 8 weeks of age. Engraftment was allowed to occur for 8 months, then engrafting macrophages and microglia were isolated from whole brains for RNA-Seq.
Project description:Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.
Project description:In this study, we demonstrate a newly derived mouse model that supports engraftment of human hematopoietic stem cells (HSCs) in the absence of irradiation. We cross the NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (NSG) strain with the C57BL/6J-Kit(W-41J)/J (C57BL/6.Kit(W41)) strain and engraft, without irradiation, the resulting NBSGW strain with human cord blood CD34+ cells. At 12-weeks postengraftment in NBSGW mice, we observe human cell chimerism in marrow (97% ± 0.4%), peripheral blood (61% ± 2%), and spleen (94% ± 2%) at levels observed with irradiation in NSG mice. We also detected a significant number of glycophorin-A-positive expressing cells in the developing NBSGW marrow. Further, the observed levels of human hematopoietic chimerism mimic those reported for both irradiated NSG and NSG-transgenic strains. This mouse model permits HSC engraftment while avoiding the complicating hematopoietic, gastrointestinal, and neurological side effects associated with irradiation and allows investigators without access to radiation to pursue engraftment studies with human HSCs.