Syngeneic Bone Marrow Cells Research Articles

Bone Marrow Transfer in Relapsing-Remitting EAE Ameliorates Disease at First Remission, with No Synergistic Effect upon Co-Transplantation with Mesenchymal Stem Cells.

Multiple sclerosis (MS) is a neurological disorder characterized by an autoimmune response, demyelinating plaques and axonal damage. Intense immunosuppression (II) followed by autologous hematopoietic stem cell transplantation has been proposed as a treatment in severe forms of MS. We have used murine relapsing-remitting (RR) experimental autoimmune encephalomyelitis (RR-EAE) to evaluate the transplantation of syngeneic bone marrow cells (BMC) after II, in combination with mesenchymal stem cells (MSCs) as a new therapeutic adjunct capable of improving immune reconstitution. In EAE-affected mice treated with BMC alone, we observed a drastic reduction in the clinical course only during the early RR phase of the disease. There was no difference in the RR-EAE clinical course between mice treated with BMC alone and co-transplanted mice. To analyze the immune reconstitution, we quantified the circulating immune cells in naïve and RR-EAE-affected mice after II, with BMC alone or in combination with MSC. Although II resulted in reduced numbers of circulating immune cells, reconstitution did not differ in co-transplanted mice. During the early phase of the disease, IL-4 was significantly elevated in co-transplanted mice, as compared to those treated with BMC alone. These data suggest that BMC transplantation after II transiently ameliorates the clinical symptoms of RR-EAE, but that co-transplantation with MSC has no synergistic effect.

Erythroid Specificity and Safety of Globin-Encoding Lentiviral Vectors

The beta-thalassemias and sickle cell disease are the most common monogenic inherited blood disorders, both arising from mutations affecting the beta-globin locus. Recent clinical trials utilizing lentiviral-mediated beta-globin gene transfer in autologous CD34+ cells have shown encouraging results in patients with non-beta-zero thalassemias and sickle cell anemia. A case report of insertional mutagenesis in a beta-thalassemia patient resulted in a prolonged clonal expansion that eventually regressed without progressing to leukemic transformation. Nonetheless, this occurrence and the need to insert multiple vector copies per cell when using globin vectors that provide insufficient globin expression to achieve curative responses from a single integrated copy per cell, raise some questions about the erythroid specificity and safety of globin vectors. We thus found it imperative to investigate globin vector expression in hematopoietic progenitors and in non-erythroid cells. To this end, we investigated what regulatory elements, including locus control region (LCR) hypersensitive sites (HS) and others, achieve the highest beta-globin expression per vector copy while at the same time minimizing non-erythroid transcriptional activity. We developed an invivo assay to track the enhancer/promoter activity of different HS elements in hematopoietic progenitor and differentiated cell subsets. We designed lentiviral vectors expressing hrGFP under the control of a short β-globin promoter (137bp) controlled by a set of LCR HS elements and/or a novel biliverdin (BLV) enhancer. Analysis of hrGFP expression in bone marrow cells derived from C57BL6 mice transplanted with transduced syngeneic bone marrow cells revealed that many of the vectors encompassing elements thought to encode erythroid-specific elements were in fact expressed in long term-HSC (LT-HSC), short term-HSC (ST-HSC), multipotent progenitors (MPP), pre-GM, Granulocyte/macrophage progenitors (GMP) and megakaryocyte progenitors (MKP). These findings confirmed that ectopically integrated erythroid regulatory elements can serve as transcriptional enhancers in non-erythroid cells. This transcriptional leakiness was confirmed in therapeutic globin vectors harboring a combination of LCR elements. To avoid these effects, we flanked the globin transcription unit with a small human enhancer-blocking element called A1, kindly provided by the late George Stamatoyannopoulos (Liu, M. et al. 2015). The insulated vector showed markedly reduced non-erythroid expression in non-erythroid cells including ST-HSC, MPP, Pre-GM and GMP. Expression in mature erythroid cells was unchanged. Our findings underscore the benefits of selecting lineage-specific regulatory elements with the least lineage promiscuity for therapeutic vectors, with the eventual addition of enhancer-blocking elements. Based on these results, we are now investigating new globin expression vectors with optimal combination of HS elements. Such novel globin vectors should minimize the risk of oncogene trans-activation in hematopoietic progenitor cells and therefore improve the safety of globin gene therapy while providing lineage-specific high-level expression. Disclosures Sadelain: Mnemo: Patents & Royalties; Fate Therapeutics: Patents & Royalties, Research Funding; Atara: Patents & Royalties, Research Funding; Takeda: Patents & Royalties, Research Funding; Minerva: Other: Biotechnologies , Patents & Royalties.

A Novel Evidence That Mannan Binding Lectin (MBL) Pathway of Complement Cascade Activation is Involved in Homing and Engraftment of Hematopoietic Stem Progenitor Cells (HSPCs)

Delayed homing and engraftment of hematopoietic stem progenitor cells (HSPCs) or even failure to engraft at all is significant clinical problem after hematopoietic transplant. Therefore, in order to develop more efficient homing and engraftment facilitating strategies it is important to learn more about this process. Our team has postulated that myeloablative conditioning for transplantation induces in bone marrow (BM) microenvironment a state of sterile inflammation in which elements of innate immunity activated by radio- or chemotherapy conditioning for transplant play an important role. In frame with this claim we reported that a significant role in this process plays activation of complement cascade (ComC). Accordingly, mice that that lack a fifth component (C5) of ComC turned out to engraft poorly with normal syngeneic BM cells as compared to normal control animals. In extension of our previous studies we provide for first time evidence that mannan binding lectin (MBL) pathway is involved in activation of ComC in myeloablated transplant recipient BM and thus plays an important role in homing and engraftment of HSPCs. To support this MBL-KO mice show significant defect in hematopoietic reconstitution after hematopoietic transplantation. This correlates with a decrease in expression of stromal derived factor-1 (SDF-1) and impaired activation of Nlrp3 inflammasome in irradiated BM of these mice.

In vitro and in vivo modulation of Hematopoietic Stem Cell (HSCs) differentiation into T and B cells by poly dispersed acid-functionalized single-walled carbon nanotubes (AF-SWCNTs)

Abstract Background AF-SWCNTs are known to have significant effect on the immune function. There is not much information available on the effect of carbon nanotubes (CNTs) on the differentiation of the HSCs. In the present study we have focused on studying the modulatory effects of AF-SWCNTs on the differentiation of HSCs into T & B cells. Methods Single-walled CNTs were acid-functionalized by treating with concentrated sulfuric & nitric acid; thus, tagged with Alexa Fluor 633 to get fluorescent AF-SWCNTs (FAF-SWCNTs). To study internalization, magnetic beads enriched C57bl/6 mouse bone marrow (BM) HSCs (Lin− Sca1+cKit+) were incubated with FAF-SWCNTs & studied using flow cytometry & confocal microscopy. HSCs were cultured with a cocktail of cytokines to induce differentiation to T & B cells, in (+/−) AF-SWCNTs & monitored by live cell imaging. For in vivo ontogenesis study, mice were lethally irradiated (gamma irradiation) & reconstituted using syngeneic BM cells pretreated (+/−) AF-SWCNTs. At different time points, appearance of T & B cells in the spleen was monitored. Results Our results indicate that HSCs internalized significant amounts of FAF-SWCNTs in vitro that remained essentially localized in cytoplasm. Differentiation of cytokines stimulated HSCs to T & B cell was significantly suppressed by AF-SWCNTs. Results of our in vivo experiments indicated that after 17th day of BM transplantation, significant numbers of T & B cells appeared in the reconstituted mouse spleen. However, when AF-SWCNTs pretreated BM cells used to reconstitute irradiated mice, significant decline in both of their percentage & absolute recovery was noted. These results indicate that CNTs may significantly modulate the differentiation of HSCs into lymphocytes.

The effect of transplantation of bone marrow cells induced by the contact with thymus-derived multipotent stromal cells on the immune system of mice, regenerating after cyclophosphamide treatment

The effect of transplantation of syngeneic bone marrow cells (BMCs) after their contact in vitro with thymus-derived multipotent stromal cells (MSCs) for regeneration of damaged by cyclophosphamide immune system of mice was studied.Materials and methods. MSCs were obtained from C57BL/6 mice’s thymus by explants method. BMCs were obtained by flushing the femurs. BMCs were induced for 2 hours on the monolayer of thymus-derived MSCs. The immune deficiency of mice was modelled using cyclophosphamide injection. After that, cell transplantation was performed and the state of the immune system was assessed. The number of erythrocytes, hematocrit, hemoglobin concentration in the peripheral blood; the phases of the cell cycle and apoptosis of mesenteric lymph node cells were determined. The amount of antibody-producing cells in the spleen and the delayed hypersensitivity response was determined. The study of proliferative and cytotoxic activity of natural killer lymphocytes, the analysis of phagocytosis, spontaneous and induced bactericidal activity of peritoneal macrophages were performed.Results. It was shown that unlike intact bone marrow cells, BMCs induced by thymus-derived MSCs provided increased spontaneous proliferative activity of lymphocytes with a decrease in the number of lymph node cells in G0/G1 phase by 6.2 % and an increase the number of lymphocytes in S+G2/M phase by 28 % in comparison with the group of mice treated with cyclophosphamide, as well as the recovery of cellularity of the bone marrow, lymph nodes and spleen. At the same time in the lymph nodes, the number of cells in the apoptosis increased. BMCs induced by MSCs showed a pronounced negative effect on natural cytotoxicity, reducing its rates by 3 times compared with the group of cyclophosphamide-treated mice, and on adaptive immunity: the rates of delayed hypersensitivity response decreased by 1.7 times, number of antibody-producing cells by 1.8 times. Red blood cell regeneration was stimulated by intact BMCs, which was manifested by the normalization of hematocrit and hemoglobin and an increase in the number of reticulocytes in the blood by 2.2 times compared with the group of mice treated with cyclophosphamide.Conclusion. Transplanted BMCs improve erythropoiesis in mice after cyclophosphamide treatment, and BMCs, previously induced by thymus-derived MSCs, lose this ability. BMCs after co-culture are strongly activated to impact on the immune system, which is most likely due to the effect of contact interaction with thymus-derived MSCs, which is known, effectively affect hematopoietic cells and possess immunomodulatory properties.

Regulatable Lentiviral Hematopoietic Stem Cell Gene Therapy in a Mouse Model of Parkinson's Disease.

Glial cell line-derived neurotrophic factor (GDNF) exhibits potent neuroprotective properties in preclinical models of Parkinson's disease (PD), but challenges in GDNF delivery have been reported from clinical trials. To address this barrier, we developed a hematopoietic stem cell transplantation-based macrophage-mediated GDNF therapy platform. Here, we introduced a regulatable lentiviral vector (LV-MSP-Tet-Off-hGDNF) to allow the expression of human GDNF (hGDNF) to be adjusted or stopped by oral administration of doxycycline (Dox). C57BL/6J mice were lethally irradiated with head protection and then transplanted with syngeneic bone marrow cells transduced with either the hGDNF-expressing vector or a corresponding GFP-expressing vector, LV-MSP-Tet-Off-GFP. Suppression of vector gene expression was achieved through administration of Dox in drinking water. To create a toxin-induced Parkinsonian model, mice were injected in two cycles with MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) to yield nigral cell/striatal dopamine loss and behavioral deficits. During the presence of Dox in the drinking water, plasma GDNF was at a basal level, whereas during the absence of Dox, plasma GDNF was significantly elevated, indicating reliable regulation of therapeutic gene expression. Midbrain GDNF levels were altered in parallel, although these did not return completely to basal levels during the periods of Dox withdrawal. Motor activities of the MPTP-Tet-off-hGDNF group were comparable to those of the Tet-off-GFP (subject to no MPTP treatment) group, but substantially better than those of the MPTP-Tet-off-GFP group. Interestingly, the improvement in motor activities was sustained during the Dox-withdrawn periods in MPTP-Tet-off-hGDNF animals. Neuroprotection by therapeutic GDNF expression was further evidenced by significant amelioration of nigral tyrosine hydroxylase loss after both the first and second MPTP treatment cycles. These data suggest that neurotrophic factor expression can be upregulated to achieve efficacy or downregulated in case of off-target effects or adverse events, a feature that may eventually increase the acceptance of this potentially neuroprotective/disease-modifying PD therapy.

Bone Marrow Transplantation after Nonmyeloablative Treosulfan Conditioning Is Curative in a Murine Model of Sickle Cell Disease

Allogeneic hematopoietic stem cell transplantation (HSCT) can be curative for patients with sickle cell disease (SCD). However, morbidity associated with myeloablative conditioning and graft-versus-host disease has limited its utility. To this end, autologous HSCT for SCD using lentiviral gene-modified bone marrow (BM) or peripheral blood stem cells has been undertaken, although toxicities of fully ablative conditioning with busulfan and incomplete engraftment have been encountered. Treosulfan, a busulfan analog with a low extramedullary toxicity profile, has been used successfully as part of a myeloablative conditioning regimen in the allogeneic setting in SCD. To further minimize toxicity of conditioning, noncytotoxic monoclonal antibodies that clear stem cells from the marrow niche, such as anti-c-Kit (ACK2), have been considered. Using a murine model of SCD, we sought to determine whether nonmyeloablative conditioning followed by transplantation with syngeneic BM cells could ameliorate the disease phenotype. Treosulfan and ACK2, in a dose-dependent manner, decreased BM cellularity and induced cytopenia in SCD mice. Conditioning with treosulfan alone at nonmyeloablative dosing (3.6 g/kg), followed by transplantation with syngeneic BM donor cells, permitted long-term mixed-donor chimerism. Level of chimerism correlated with improvement in hematologic parameters, normalization of urine osmolality, and improvement in liver and spleen pathology. Addition of ACK2 to treosulfan conditioning did not enhance engraftment. Our data suggests that pretransplant conditioning with treosulfan alone may allow sufficient erythroid engraftment to reverse manifestations of SCD, with clinical application as a preparative regimen in SCD patients undergoing gene-modified autologous HSCT.

Chimerism and gene therapy - Lessons learned from non-conditioned murine bone marrow transplantation models.

Hematopoietic stem and progenitor cells (HSPCs) can be used as a vector for gene therapies. In order to predict the number of HSPCs cells necessary to achieve the target level of chimerism in an autologous setting, syngeneic male bone marrow (BM) cells were transplanted into 35 non-conditioned female BALB/c mice. The resulting chimerism was determined at 6-53weeks using qPCR, cell subpopulation sorting, and colony-forming units (CFU) analysis. After the transplantation of 125.8±2.5million nucleated BM cells, the BM of recipients contained 20.0±2.8% donor cells, representing a chimerism of 0.16±0.02% per one million transplanted nucleated BM cells. Chimerism levels in the BM, neutrophils, and Bcells were comparable, whereas in Tcells it was lower, and in CFU was approximately twice greater than in BM. By extrapolating our murine data, and data from some previous studies to a human non-conditioned autologous CD34+ HSPC transplantation setting, we conclude that approximately 44million CD34+ HSPCs would be needed to achieve 20% donor chimerism in a 70-kg human, which could serve as a starting point for the future use of HSCPs in gene and cell therapy.

Vascular Alterations in a Murine Model of Acute Graft-Versus-Host Disease Are Associated with Decreased Serum Levels of Adiponectin and an Increased Activity and Vascular Expression of Indoleamine 2,3-Dioxygenase.

Graft-versus-host disease (GVHD) is the limiting complication after bone marrow transplantation (BMT), and its pathophysiology seems to be highly influenced by vascular factors. Our study aimed at elucidating possible mechanisms involved in vascular GVHD. For this purpose, we used a fully MHC-mismatched model of BALB/c mice conditioned according to two different intensity protocols with total body irradiation and transplantation of allogeneic (C57BL/6) or syngeneic bone marrow cells and splenocytes. Mesenteric resistance arteries were studied in a pressurized myograph. We also quantified the expression of indoleamine 2,3-dioxygenase (IDO), endothelial (eNOS), and inducible NO synthase (iNOS), as well as several pro- and anti-inflammatory cytokines. We measured the serum levels of tryptophan (trp) and kynurenine (kyn), the kyn/trp ratio (KTR) as a marker of IDO activity, and adiponectin (APN). The myographic study showed a correlation of GVHD severity after allogeneic BMT with functional vessel alterations that started with increased vessel stress and ended in eccentric vessel remodeling, increased vessel strain, and endothelial dysfunction. These alterations were accompanied by increasing IDO activity and decreasing APN levels in the serum of allogeneic animals. The mRNA expression showed significantly elevated IDO, decreased eNOS, and elevation of most studied pro- and anti-inflammatory cytokines. Our study provides further data supporting the importance of vessel alterations in GVHD and is the first to show an association of vascular GVHD with hypoadiponectinemia and an increased activity and vascular expression of IDO. Whether there is also a causative involvement of these two factors in the development of GVHD needs to be further investigated.

Cytosine-Phosphorothionate-Guanine Oligodeoxynucleotides Exacerbates Hemophagocytosis by Inducing Tumor Necrosis Factor–Alpha Production in Mice after Bone Marrow Transplantation

Hemophagocytic syndrome (HPS) is frequently associated with hematopoietic stem cell transplantation and is treated with some benefit derived from TNF-α inhibitors. However, the mechanisms of how HPS occurs and how a TNF-α inhibitor exerts some benefit to HPS management have remained unclear. We evaluated the effect of toll-like receptor (TLR) ligands, especially focusing on cytosine-phosphorothionate-guanine oligodeoxynucleotide (CpG), a TLR9 ligand, on HPS in mice that underwent transplantation with syngeneic or allogeneic bone marrow (BM) cells (Syn-BMT, Allo-BMT), or with allogeneic BM cells plus splenocytes to promote graft-versus-host disease (GVHD mice). Hemophagocytosis was a common feature early after all BMT, but it subsided in Syn-BMT and Allo-BMT mice. In GVHD mice, however, hemophagocytosis persisted and was accompanied by upregulated production of IFN-γ but not TNF-α, and it was suppressed by blockade of IFN-γ but not TNF-α. A single injection of the TLR9 ligand CpG promoted HPS in all BMT mice and was lethal in GVHD mice, accompanied by greatly upregulated production of TNF-α, IL-6, and IFN-γ. Blocking of TNF-α, but not IL-6 or IFN-γ, suppressed CpG-induced HPS in all BMT mice and rescued GVHD mice from CpG-induced mortality. Thus, TLR9 signaling mediates TNF-α–driven HPS in BMT mice and is effectively treated through TNF-α inhibition.

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic, but Not Syngeneic Recipient Mice

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.

Developing a Mouse Model to Study Mesenchymal Stem Cell Survival, Proliferation, and Migration in Lumbar Posterolateral Spinal Fusion

Introduction While spinal fusion surgery has advanced over the past decade, a great deal is unknown regarding the underlying mechanisms that contribute to spinal fusion failure or pseudoarthrosis. Clinically, bone marrow aspirate has been successfully used alongside a variety of grafting materials to augment fusion (Neen et al. Spine (Phila Pa 1976). 2006;31(18):E636; Carter et al. Spine J. 2009;9(6):434). To date, studies evaluating these procedures have focused on radiographic monitoring and endpoint analysis. Little is known about the survival and proliferation of these transplanted mesenchymal cells or how much they contribute to bone formation compared with host cells from adjacent decorticated bone. Insights into these cell dynamics will give us a deeper understanding of the biological processes underlying spinal fusion and allow for evaluation and development of fusion therapies that reduce rates of pseudoarthrosis. Material and Methods A total of 40 FVB/NJ mice underwent posterolateral lumbar spinal fusion surgery and were divided into four groups based upon the fusion graft employed: syngeneic iliac crest bone graft ( n = 10), Vitoss bone-matrix alone ( n = 10), Vitoss bone-matrix with syngeneic bone marrow cells (2.5 × 106 cell/side) ( n = 10), and Vitoss bone-matrix with transgenic luciferase-expressing syngeneic bone marrow cells (2.5 × 106 cell/side) ( n = 10). Bone marrow cells were isolated from the long bones of either syngeneic FVB/NJ mice or syngeneic FVB mice which constitutively express the luciferase gene (FVB-Tg (CAG-luc,-GFP) L2G85Chco/J mice). The in vivo survival, distribution, and proliferation of the luciferase-expressing bone marrow-derived cells were monitored via bioluminescence imaging over a period of 8 weeks. Spinal fusion was assessed 4, 6, and 8 weeks postsurgery via micro-CT imaging, as well as via manual palpation at 8 weeks. Immunohistochemistry was performed to assess fusion bone growth as well as determine the number of donor versus host cells within the fusion mass. Results Bioluminescent imaging data demonstrated quantifiable cellular viability within the fusion mass as early as 24 hours following surgery, with increasing bioluminescent signal over the first 2 weeks postsurgery, thus indicating cellular proliferation. Bioluminescence decreased between weeks 4 and 8 suggesting a loss of transplanted mesenchymal progenitor cells within the fusion mass at later time points. Correlations with histology and micro-CT imaging of the developing fusion mass are currently under analysis. Conclusions We have demonstrated a novel murine model which enables noninvasive in vivo monitoring of implanted transgenic luciferase-expressing bone marrow cells following spinal fusion surgery. Future applications of this model will include the assessment of additional therapeutics for improving spinal fusion in translational research studies.

Uterine-derived progenitor cells are immunoprivileged and effectively improve cardiac regeneration when used for cell therapy

Cell therapy to prevent cardiac dysfunction after myocardial infarction (MI) is less effective in aged patients because aged cells have decreased regenerative capacity. Allogeneic transplanted stem cells (SCs) from young donors are usually rejected. Maintaining transplanted SC immunoprivilege may dramatically improve regenerative outcomes. The uterus has distinct immune characteristics, and we showed that reparative uterine SCs home to the myocardium post-MI. Here, we identify immunoprivileged uterine SCs and assess their effects on cardiac regeneration after allogeneic transplantation. We found more than 20% of cells in the mouse uterus have undetectable MHC I expression by flow cytometry. Uterine MHC I((neg)) and MHC I((pos)) cells were separated by magnetic cell sorting. The MHC I((neg)) population expressed the SC markers CD34, Sca-1 and CD90, but did not express MHC II or c-kit. In vitro, MHC I((neg)) and ((pos)) SCs show colony formation and endothelial differentiation capacity. In mixed leukocyte co-culture, MHC I((neg)) cells showed reduced cell death and leukocyte proliferation compared to MHC I((pos)) cells. MHC I((neg)) and ((pos)) cells had significantly greater angiogenic capacity than mesenchymal stem cells. The benefits of intramyocardial injection of allogeneic MHC I((neg)) cells after MI were comparable to syngeneic bone marrow cell transplantation, with engraftment in cardiac tissue and limited recruitment of CD4 and CD8 cells up to 21 days post-MI. MHC I((neg)) cells preserved cardiac function, decreased infarct size and improved regeneration post-MI. This new source of immunoprivileged cells can induce neovascularization and could be used as allogeneic cell therapy for regenerative medicine.

Preclinical evaluation of efficacy and safety of an improved lentiviral vector for the treatment of β-thalassemia and sickle cell disease.

A previously published clinical trial demonstrated the benefit of autologous CD34+ cells transduced with a self-inactivating lentiviral vector (HPV569) containing an engineered β-globin gene (βA-T87Q-globin) in a subject with β-thalassemia major. This vector has been modified to increase transduction efficacy without compromising safety. In vitro analyses indicated that the changes resulted in both increased vector titers (3 to 4 fold) and increased transduction efficacy (2 to 3 fold). An in vivo study in which 58 β-thalassemic mice were transplanted with vector- or mock-transduced syngenic bone marrow cells indicated sustained therapeutic efficacy. Secondary transplantations involving 108 recipients were performed to evaluate long-term safety. The six month study showed no hematological or biochemical toxicity. Integration site (IS) profile revealed an oligo/polyclonal hematopoietic reconstitution in the primary transplants and reduced clonality in secondary transplants. Tumor cells were detected in the secondary transplant mice in all treatment groups (including the control group), without statistical differences in the tumor incidence. Immunohistochemistry and quantitative PCR demonstrated that tumor cells were not derived from transduced donor cells. This comprehensive efficacy and safety data provided the basis for initiating two clinical trials with this second generation vector (BB305) in Europe and in the USA in patients with β-thalassemia major and sickle cell disease.

Incidence of bone formation by whole bone marrow cell suspensions and by its stromal cell cultures injected into kidney parenchyma of Balb/c mice.

The evaluation of incidence of bone formation by whole syngeneic bone marrow cell suspension and by bone marrow stromal cell cultured in vitro injection into kidney parenchyma was done. Bone tissue was found in 26 kidneys out of 100 injected with whole bone marrow cells suspension. Cultured stromal bone marrow cells grafted into kidney parenchyma produced ossicles in only 4 out of 101 injected kidneys. Such low incidence of bone forming ability of the marrow stromal cell cultures grafted into kidney indicate their useless for study on bone histogenesis in the kidney by murine marrow stromal cell cultures.

Angptl-4 Is Upregulated Under Inflammatory Conditions In The BM Of Mice, Expands Myeloid Progenitors and Accelerates Reconstitution Of Platelets After Myelosuppressive Therapy

The concerted action of hematopoiesis supporting cytokines such as G-CSF, GM-CSF or IL-6 regulates hematopoiesis during steady state and emergency situations. Respective knockout mice show defects both in production and function of myelopoietic effector cells. However, alternative pathways are likely to exist as mice with single or combined deficiencies for G-CSF, GM-CSF, and IL-6 or G-CSF and GM-CSF are still able to mount reactive neutrophilia responses during inflammatory conditions. In order to identify pathways for inflammation induced enhancement of hematopoiesis as well as to find new cytokines, which enhance myeloid cell regeneration, we analyzed the bone marrow (BM) of lipopolysaccharide (LPS) and vehicle injected wild type (WT) mice (single IP- injection) by gene expression microarray. Focusing on the identification of genes encoding for secreted or membrane proteins, we found 83 candidates to be up- and 14 to be downregulated after LPS treatment.Among known candiates, we found angiopoietin-like 4 (Angptl-4) as a predominantly upregulated gene in the BM of LPS-treated WT-mice. Upregulation was confirmed by RT-PCR as well as by Elisa in the BM of LPS treated mice and bone marrow stromal cells (BMSC) were identified as candidate producer cells. Functionally, we found recombinant Angptl-4 to stimulate the proliferation of myeloid colony-forming units (CFU) in vitro. In mice, repeated injections of Angptl-4 increased BM progenitor cell frequency and this was paralleled by a relative increase in phenotypically defined granulocyte-macrophage progenitors (GMPs). Furthermore, in vivo treatment with Angptl-4 resulted in elevated platelet counts both in untreated animals and after myelosuppressive therapy. After lethal irradiation and transplantation of syngeneic BM cells repetitive injections of recombinant Angptl-4 for 5 consecutive days resulted in an accelerated reconstitution of platelets starting at day 8 after transplantation. The 50% pre-treatment platelet count was reached on day 14 in Angptl-4-treated animals as compared to day 21 for transplanted controls receiving no Angptl-4 (n=8; p=0.03, student´s T test). In contrast, transplantation of BM cells from Angptl-4 pre-treated donor mice had no effect on the recovery of platelets in this setting. The frequency of CD41lowCD61+ immature megakaryocytes was significantly increased in the BM of Angptl-4 injected as compared to control mice (27% vs 19% of total megakaryocytes; p= 0.008, student´s T test). Furthermore, bone marrow cytology revealed local accumulation of megakaryocytes carrying dysplastic features in Angptl-4 injected mice.In summary, our data suggest that Angptl-4 plays a complementary role on hematopoiesis during emergency situations like sepsis. The use of Angptl-4 in the setting of autologous stem cell transplantation could represent a potential approach to accelerate the reconstitution of megakaryopoiesis. Disclosures:No relevant conflicts of interest to declare.

The Role of Programmed Cell Death Ligand-1 (PD-L1/CD274) in the Development of Graft versus Host Disease

Programmed cell death ligand-1 (PD-L1/CD274) is an immunomodulatory molecule involved in cancer and complications of bone marrow transplantation, such as graft rejection and graft-versus-host disease. The present study was designed to assess the dynamic expression of this molecule after hematopoietic stem cell transplantation in relation to acute graft-versus-host disease. Female BALB/c mice were conditioned with busulfan and cyclophosphamide and transplanted with either syngeneic or allogeneic (male C57BL/6 mice) bone marrow and splenic cells. The expression of PD-L1 was evaluated at different time points employing qPCR, western blot and immunohistochemistry. Allogeneic- but not syngeneic-transplanted animals exhibited a marked up-regulation of PD-L1 expression in the muscle and kidney, but not the liver, at days 5 and 7 post transplantation. In mice transplanted with allogeneic bone marrow cells, the enhanced expression of PD-L1 was associated with high serum levels of IFNγ and TNFα at corresponding intervals. Our findings demonstrate that PD-L1 is differently induced and expressed after allogeneic transplantation than it is after syngeneic transplantation, and that it is in favor of target rather than non-target organs at the early stages of acute graft-versus-host disease. This is the first study to correlate the dynamics of PD-L1 at the gene-, protein- and activity levels with the early development of acute graft-versus-host disease. Our results suggest that the higher expression of PD-L1 in the muscle and kidney (non-target tissues) plays a protective role in skeletal muscle during acute graft-versus-host disease.

Anti-CD45 Radioimmunotherapy Using the Alpha-Emitting Radionuclide 211At Combined with Bone Marrow Transplantation Prolongs Survival in a Disseminated Murine Leukemia Model

AbstractAbstract 4096 Background:Despite aggressive hematopoietic cell transplant (HCT) strategies, many patients with acute myeloid leukemia (AML) relapse. Our group has explored radioimmunotherapy (RIT) using anti-CD45 antibodies (Ab) labeled with β-emitting radionuclides such as 131I and 90Y as a means to augment cytotoxicity and reduce relapse. This approach has been limited by their low energy levels (0.66–2.3 MeV) and potential non-specific toxicities due to their relatively long path lengths (0.3–2.3 mm). Conversely, α-emitting agents display higher energy levels (8 MeV) delivered over a short path-length (∼60–80 μm) that can lead to superior therapeutic ratios of absorbed radiation doses that may reduce AML relapse. For this purpose 211At is an α-emitter with an attractive half-life (7.2 hours), energy profile (6.8 MeV averages of two alpha decays, 5.9 and 7.5 MeV) and path length (average range 55–70 μm). Therefore, we evaluated the efficacy and toxicity of anti-CD45 RIT using 211At in a clinically relevant CD45+disseminated murine leukemia model. Methods:SJL/J mice were given 105 syngeneic SJL leukemic peripheral blood mononuclear cells (PBMCs) via tail vein, followed two days later by211At-labeled anti-murine CD45 Ab-decaborate(2-) conjugate (30F11-B10) or 211At-labeled negative control Ab-decaborate(2-) conjugate (rat IgG-B10) in tissue biodistribution studies. Groups of 5 mice were euthanized 6, 24 and 48 hours later and organs were harvested and analyzed in a gamma counter to yield percent of the injected dose per gram (% ID/g). To assess toxicities associated with this approach, SJL/J mice were treated with 12–24 μCi of 211At-30F11-B10 and then evaluated weekly thereafter for impact on blood counts, as well as changes in hepatic and renal function. In HCT therapeutic studies, groups of 10 leukemic mice per dose were injected with 12–24 μCi of 211At-30F11-B10 or 211At-rat Ab-B10, followed two days later by rescue with 15 × 106 syngeneic bone marrow (BM) cells. Results:Delivery of 211At-30F1-B10 demonstrated excellent localization to the BM and spleen at 24 hours (79 and 18% ID/g, respectively) post injection with lower kidney and lung uptake (8.4 and 8.3% ID/g, respectively) at the same time point. Anti-CD45 RIT using 211At-30F11-B10 followed by syngeneic HCT led to a dose-dependent survival benefit in leukemic mice with a median survival (OS) of 120, 98 days, and 62 days for animals treated with 24, 20, and 12 μCi 211At-30F11-B10, respectively, compared with untreated control mice (median OS of 36 days) and mice treated with non-specific 211At-labeled rat Ab-B10 (median OS of 46 days) (Figure). Moreover, anti-CD45 RIT with 211At-30F11-B10 led to minimal toxicity with mild dose dependent leukopenia as the most pronounced lab abnormality. White blood cell count nadir was between 2.5 and 4.2 k/μL two weeks after HCT for mice treated with 24 and 12 μCi 211At-30F11-B10, respectively. Counts recovered to normal levels (6–8 K/μL) 4 weeks after HCT. Mild increases in transaminase levels were seen in mice that received 211At-30F11-B10, yet these values remained within the normal ranges (ALT 68–75 IU/L; AST 155–170 IU/L). Renal function after 211At-30F11-B10 did not significantly deviate from baseline (BUN 15–17 mg/dL; Cr 0.3–0.4 mg/dL). Conclusion:Taken together, these data suggest that anti-CD45 RIT using the α-emitting radionuclide 211At in conjunction with HCT is a promising therapeutic option for AML. Excellent targeting of radiation doses to BM and spleen was demonstrated with a favorable toxicity profile. Further investigation of anti-CD45 RIT for AML using 211At in clinical trials appears to be warranted. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Preconditioning with AMD3100 Mobilization Promotes Homing and Long-Term Engraftment of Hematopoietic Stem Cells During Syngeneic Transplantation in the Murine Model

Abstract 1886 IntroductionTransplantation efficacy can be limited by inadequate hematopoietic stem cell (HSC) number, homing, engraftment, or self-renewal. Traditional conditioning prior to HSC transplantation by myeloablative chemotherapy with or without radiation provides therapeutic benefit but also results in immunosuppression and tissue damage that contribute to increased morbidity and mortality, therefore, development of safe myeloablative strategies is a clinical priority. AMD3100 has been shown to be a potent mobilizer of HSCs in multiple animal species and humans and acts by specifically blocking the CXCR4/CXCL12 (SDF-1α) axis that is critical for HSC homing and retention in the niche. Therefore, we hypothesized that rapid mobilization of host HSC out of the bone marrow would result in increased availability of niches for subsequently infused donor HSCs. MethodsC57/BL6 mice were injected subcutaneously with 5 mg/kg AMD3100 and at indicated time points, 40×106 C57/BL6 GFP+ donor bone marrow mononuclear cells were intravenously delivered. The control group received injection of PBS. For additive treatment of the donor cells with PGE2 and Diprotin A, B6 GFP+ HSCs were treated with 1μM PGE2 or 5 mM Diprotin A and then delivered into the recipient mice 60 min post-AMD3100 administration. For combinatorial treatment with both drugs, the cells were treated with PGE2 for 2 hours at 4 °C and then treated with Diprotin A at RT for 15 min prior to transplantation. For homing studies, twelve hours after transplantation, the mononuclear cells from peripheral blood and bone marrow were harvested for flow cytometry and CFU-C assay. Fold increases were based on comparison with control mice that received PBS injection and DMEM-treated donor cells. For long-term engraftment studies, the assays were performed four months after transplantation. ResultsThe results demonstrated that a single administration of AMD3100 resulted in significantly increased availability of bone marrow niches by mobilizing the host HSCs into the periphery in a time-dependent manner. The maximal effect of up to 90% vacancy of the niche was observed at 1 hour after AMD3100 administration. Infusion of syngeneic donor bone marrow cells between 45 and 60 min post-AMD3100 administration resulted in maximum homing and engraftment efficiency. Donor CXCR4+CD49d+ cells preferentially homed to the host bone marrow niche. Pretreatment of donor HSCs with PGE2 or Diprotin A (CD26 blockade) further enhanced the homing efficiency following AMD3100 preconditioning. Moreover, AMD3100 preconditioning with or without PGE2 or Diprotin A also resulted in long-term engraftment of donor HSCs. The occupancy of the marrow niche by HSCs was restored to the full capacity in the long term, indicating treatment with these drugs did not result in toxicity. PGE2 and Diprotin A pre-treatment, each of which have been demonstrated to selectively improve donor cell homing and engraftment, further enhances the positive effects of AMD3100 conditioning alone. ConclusionOur data support the efficacy of AMD3100 mobilization, alone or in combination with donor cell pre-treatment strategies, as a safe-and-effective preconditioning method for hematopoietic stem cell transplantation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Ischemia induces P-selectin-mediated selective progenitor cell engraftment in the isolated-perfused heart

Clinical trials infusing Bone Marrow Cells (BMCs) into injured hearts have produced measureable improvements in cardiac performance, but were insufficient to improve patient outcomes. Low engraftment rates are cited as probable contributor to limited improvements. To understand the mechanisms that control myocardial engraftment of BMCs following ischemia-reperfusion injury, in isolated–perfused mouse hearts, stop-flow ischemia was followed by variable-duration reperfusion (0–60min) before addition of labeled syngenic BMCs to the perfusate. After a buffer-only wash, the heart was disaggregated. Retained BMCs (digest) and infused BMCs (aliquot) were compared by flow cytometry for c-kit and CD45 expression to determine the proportion of cell subtypes engrafted versus delivered (selectivity ratio). In these studies, a time-dependent selective retention of c-kit+ cells was apparent starting at 30min of reperfusion, at which time c-kit+/CD45+ BMCs showed a selectivity ratio of 18±2 (versus 2±1 in sham-ischemic controls). To study the underlying mechanism for this selective retention, neutralizing antibodies for P-selectin or L-selectin were infused into the heart preparation and incubated with BMCs prior to BMC infusion. Blocking P-selectin in ischemic hearts ablated selectivity for c-kit+/CD45+ BMCs at 30min reperfusion (selectivity ratio of 3±1) while selectivity persisted in the presence of L-selectin neutralization (selectivity ratio of 17±2). To corroborate this finding, a parallel plate flow chamber was used to study capture and rolling dynamics of purified c-kit+ versus c-kit- BMCs on various selectin molecules. C-kit+ BMCs interacted weakly with L-selectin substrates (0.03±0.01% adhered) but adhered strongly to P-selectin (0.28±0.04% adhered). C-kit- BMCs showed intermediate binding regardless of substrate (0.18±0.04% adhered on L-selectin versus 0.17±0.04% adhered on P-selectin). Myocardial ischemia–reperfusion stress induces selective engraftment of c-kit+ bone marrow progenitor cells via P-selectin activation.