Increase In Engraftment Research Articles

Fatty Bone Marrow Positively Selects Pre-Leukemic HSPCs with a DNMT3A-mutation

With aging, humans accumulate preleukemic mutations (pLMs) in hematopoietic stem and progenitor cells (HSPCs) which was termed age-related clonal hematopoiesis (ARCH). To gain a better insight changes in the HSPCs-environment crosstalk upon aging that might contribute to ARCH, it is critical to develop a multilayer perspective that integrates information on mutations, epigenetics the cellular context and the bone marrow (BM) microenvironment, since all these layers are changing during ageing. Accordingly, a key question in the field is how the ageing BM microenvironment influences clonal expansion of HSPCs. Fatty bone marrow (FBM) is one of the environmental factors that may influence clonal hematopoiesis (CH) with age. As we age, our bone marrow shifts from red to adipocyte-enriched yellow BM. We hypothesize that age related BMF accumulation may provide a selective advantage to specific pre-leukemic stem and progenitor cells (preL-HSPCs) carrying pLM. To support this hypothesis, we established a FBM model in NSG mice to enable the study of both human and rodent preL-HSPCs. Transplantation of primary human preL-HSPCs from AML patients (DNMT3A, NPM1 mutations ) into FBM resulted in enhanced engraftment compared to control mice without FBM. We further demonstrate that DNMT3A-R882H+/- mice derived BM HSPCs, engrafted significantly higher in NSG mice with FBM compared to controls. Interestingly, when DNMT3A-R882H+/- derived BM cells from middle-aged mice (12-month old) were injected into FBM mice, engraftment increased tenfold. Secondary engraftment of aged DNMT3A-R882H +/-BM derived cells resulted in an increase in engraftment upon transplantation into to FBM, suggesting enhanced in vivo self-renewal capacity of HSPCs in FBM.To study the underlying molecular mechanisms provided by the FBM to preL-HSPCs carrying DNMT3A-R882H +/-, we used a multiplex cytokine assay. In this approach we analyzed 17 common cytokines in BM following transplantation of young, two-month old, or middle-aged, 12-month old, DNMT3A-R882H +/-or control - BM derived cells into FBM. Our results show that transplanting two months old, middle-aged DNMT3A-R882H +/-or control BM derived cells to FBM resulted in a significant increase in BM mIL-6 secretion when compared to transplants into control, non-FBM mice. mIL-6 was secreted by adipocytes following irradiation regardless of which cells are transplanted. We then transplanted middle-aged DNMT3A-R882H+/- BM derived cells to FBM mice that had been treated intraperitoneally with a neutralizing IL-6 Ab. The administration of neutralizing IL-6 Ab resulted in a significant decrease in engraftment of DNMT3A-R882H+/- BM derived cells, confirming that IL6 contributes to the expansion of the DNMT3a-R882H+/- cells in FBM.In summary, these results demonstrate for the first time that the FBM provides a selective advantage to pre-leukemic cells carrying DNMT3A-R882H. Importantly, we show that IL-6 is a one of the major players in the molecular mechanism that confers the FBM advantage specifically to preL-HSPCs carrying R882H both in vitro and in vivo. DisclosuresMueller-Tidow: Janssen Cilag: Consultancy, Research Funding; Bioline: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding.

Aryl Hydrocarbon Receptor Antagonists Expand Adult Hematopoietic Stem Cells from Mobilized Peripheral Blood and Bone Marrow and Increase the Dose of CRISPR/Cas9 Gene-Edited NSG-Repopulating Cells

Background. Gene modified hematopoietic stem cell (HSC) transplant is emerging as a promising approach to treat a variety of inherited genetic diseases, but has been hampered by the inability to generate a sufficient dose of gene modified HSCs and the requirement for toxic myeloablative conditioning protocols to achieve engraftment. Ex vivo expansion of gene-modified HSCs is a promising approach to circumvent these challenges as high doses of gene modified cells have the potential to achieve robust engraftment and reduce the requirement for myeloablative conditioning. Several small molecule approaches (SR1, UM171, and HDAC inhibitors) have been reported to expand cord blood (CB) derived HSCs ex vivo but the ability of these agents to expand gene modified adult HSCs is not known. Experiments comparing these approaches for CB expansion has been reported (see Goncalves et al “Phenotype Does Not Always Equal Function: HDAC inhibitors and UM171, but not SR1, Lead to Rapid Upregulation of CD90 on Non-Engrafting CD34+CD90-negative Human Cells”) and found that aryl hydrocarbon receptor (AHR) antagonism is the most effective at expanding NSG-engrafting cells. In a clinical trial of patients receiving MGTA-456, an AHR antagonist-expanded CB CD34+ cell product, engraftment occurred at a median of 14.5 days (r 7-23), which was significantly better than identically-treated historical controls (n=151, p<0.01) (Wagner et al, Cell Stem Cell, 2016). Because of the encouraging clinical results in CB, we identified novel AHR antagonists and evaluated the ability of SR1 and the proprietary AHR antagonists to increase the number of gene modified NSG engrafting cells manufactured from mobilized peripheral blood (mPB) and bone marrow (BM) CD34+ cells.Methods. Novel, small molecules, SR1, HDAC inhibitors, and UM171 were evaluated in the presence of cytokines to expand primary human CD34+ cells ex vivo . Cell number and immunophenotype were assessed by flow cytometry, and HSC function was evaluated by cell and molecular assays in vitro . The expanded cells were transplanted into sub-lethally irradiated NSG mice to evaluate engraftment potential in vivo . For editing studies, mPB and BM CD34+ cells were electroporated with CRISPR/Cas9 RNPs targeting the beta-2 microglobulin (B2M) cell surface protein. Editing rates were evaluated by flow cytometry based on loss of protein expression and TIDE analysis. Edited cells were expanded in the presence of AHR antagonist or vehicle and transplanted into NSG mice. Engraftment and editing rates were evaluated by flow cytometry of the peripheral blood and bone marrow.Results. Based on our results, cultures expanded with an AHR antagonist showed the largest improvement in NSG engraftment levels compared to unmanipulated cells. Culture of CD34+ cells with SR1 or a novel AHR antagonist led to a 6-fold increase in CD34+ number and a significant increase in engraftment in NSG mice relative to vehicle-cultured CB derived CD34+ cells. Our proprietary compound displayed complete AHR antagonism in the dioxin response element luciferase reporter assay and was a more potent antagonist compared to SR1 (a 12-fold increase in potency). To evaluate the ability of the AHR antagonist compound to effectively expand gene edited cells, mPB and BM derived CD34+ cells were treated with either vehicle or AHR antagonist and the following day edited with CRISPR/Cas9 RNPs targeting B2M. Following 7 days of expansion, the vehicle or AHR antagonist-treated cells showed 87% and 84% loss of the target protein, respectively. The expanded culture contained 3.4-fold more CD34+CD90+ cells than the vehicle-treated cells. Upon transplant, mice receiving the expanded cells showed greater than 2-fold increase in engraftment compared to those receiving vehicle-treated cells. Importantly, the editing rates of the expanded cells are maintained in vivo with an average of >75% of the human cells in the periphery of the mice showing loss of target protein.Conclusions. We have demonstrated that AHR antagonism is an effective strategy to expand functional HSCs and that novel small molecules inhibiting AHR can expand gene modified HSCs from mPB and BM. Studies are ongoing to confirm expansion with our proprietary compound of HSCs transduced with lentiviral vectors also leads to improved engraftment. This approach is a promising strategy to improve autologous gene therapy and gene editing via ex vivo HSC expansion. DisclosuresHoban:Magenta Therapeutics: Employment, Equity Ownership. Goncalves:Magenta Therapeutics: Employment, Equity Ownership. Proctor:Magenta Therapeutics: Employment, Equity Ownership. Adams:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. George:Magenta Therapeutics: Employment. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Enhanced Engraftment of Hematopoietic Progenitor Cells in Mice Treated With Granulocyte Colony-Stimulating Factor Before Low-Dose Irradiation: Implications for Gene Therapy

Gene therapy for inherited disorders of blood cells will require both efficient methods for stable gene transfer and nonablative bone marrow conditioning regimens to allow engraftment of modified hematopoietic progenitor cells (HPCs). We have used a sensitive murine system for detecting HPC engraftment using congenic C57BL/6 mice that differ at the Ly5 locus, which encodes the leukocyte common antigen. The system relies on the ability of monoclonal antibodies with specificity for Ly5.1 and Ly5.2 (revised nomenclature: CD45.1 and CD45.2, respectively) to distinguish donor and recipient peripheral blood leukocytes after transplantation of purified Sca-1+ bone marrow-derived HPCs. No detectable engraftment occurred in nonirradiated recipient mice, even when as many as 2.0 x 10(6) SCa-1+HPCs were transplanted. However, in mice receiving total body irradiation (TBI), engraftment increased as a function of pretransplantation radiation dose, number of transplanted cells, and time after transplantation. Moreover, mice receiving either granulocyte colony-stimulating factor (G-CSF) or G-CSF+ stem cell factor before low-dose TBI (160 cGy) exhibited a marked increase in engraftment compared with mice receiving a vehicle control before low-dose TBI (18.9% and 20.6% v 5.6% at a 1 month, respectively; 29% and 35% v 15.1% at 4 months, respectively). Use of growth factor pretreatment even allowed TBI doses as low as 30, 70, or 120 cGy to achieve significant engraftment of donor progenitors (0.3%, 1.5%, and 6.8% at 1 month, respectively; 1.7%, 5.8%, and 13.9% at 4 months, respectively). All animals remained healthy during the observation periods. Thus, growth factor preconditioning of the recipient followed by low-dose TBI may provide an optimal balance between safety and efficacy in achieving required levels of engraftment for gene therapy of blood disorders.

Hepatic thermal injury promotes colorectal cancer engraftment in C57/black 6 mice.

Treatment options for liver metastases (primarily colorectal cancer) are limited by high recurrence rates and persistent tumor progression. Surgical approaches to management of these metastases typically use heat energy including electrocautery, argon beam coagulation, thermal ablation of surgical margins for hemostasis, and preemptive thermal ablation to prevent bleeding or to effect tumor destruction. Based on high rates of local recurrence, these studies assess whether local effects of hepatic thermal injury (HTI) might contribute to poor outcomes by promoting a hepatic microenvironment favorable for tumor engraftment or progression due to induction of procancer cytokines and deleterious immune infiltrates at the site of thermal injury. To test this hypothesis, an immunocompetent mouse model was developed wherein HTI was combined with concomitant intrasplenic injection of cells from a well-characterized MC38 colon carcinoma cell line. In this model, HTI resulted in a significant increase in engraftment and progression of MC38 tumors at the site of thermal injury. Furthermore, there were local increases in expression of messenger ribonucleic acid (mRNA) for hypoxia-inducible factor-1α (HIF1α), arginase-1, and vascular endothelial growth factor α and activation changes in recruited macrophages at the HTI site but not in untreated liver tissue. Inhibition of HIF1α following HTI significantly reduced discreet hepatic tumor development (P = 0.03). Taken together, these findings demonstrate that HTI creates a favorable local environment that is associated with protumorigenic activation of macrophages and implantation of circulating tumors. Discrete targeting of HIF1α signaling or inhibiting macrophages offers potential strategies for improving the outcome of surgical management of hepatic metastases where HTI is used.

Mgta-145, in Combination with Plerixafor in a Phase 1 Clinical Trial, Mobilizes Large Numbers of Human Hematopoietic Stem Cells and a Graft with Immunosuppressive Effects for Allogeneic Transplant

Background . The majority of hematopoietic stem cell (HSC) transplants are performed using peripheral blood mobilized with granulocyte-colony stimulating factor (G-CSF) given over 5 days. The goal of a successful transplant is to reliably mobilize optimal numbers of HSCs necessary for rapid and consistent multilineage engraftment. Infusion of mobilized allogeneic grafts results in significant acute and chronic graft-versus-host disease (GvHD) in up to 80% of allogeneic transplant recipients. A reliable and rapid method to mobilize HSC-rich grafts with reduced GvHD potential would be clinically meaningful. In a Phase 1 study of normal volunteers, MGTA-145 (GroβT), a CXCR2 agonist, when combined with plerixafor, a CXCR4 inhibitor, robustly and rapidly mobilized sufficient HSCs for a safe transplant after only a single day of dosing and apheresis/collection. Here, we phenotypically and functionally profile these mobilized grafts obtained from human volunteers and show that MGTA-145 + plerixafor mobilizes grafts with &amp;gt;10-fold higher engraftment potential (as measured by SCID-repopulating units in NSG mice), a marked reduction in xenogeneic GvHD, and enhanced overall survival compared to G-CSF or plerixafor alone grafts. Results . In healthy donors, a peak of 40 CD34+ cells/μL were mobilized with MGTA-145 + plerixafor (n=12 donors). 11 of 12 (92%) of these donors mobilized &amp;gt;20 CD34+ cells/μL with single day dosing compared to only 8 of 14 (57%) achieving the same CD34+ cell target treated with plerixafor alone. Eight donors were mobilized with a single dose of MGTA-145 + plerixafor and apheresed on the same day. A median of 4x106 (1.5-7.0x106) CD34+ cells/kg were obtained (n=8 donors) from a median 20 (13-20) L collection. 35.8 (18.5-40.9)% of these cells were CD90+CD45RA-, a CD34+ subset enriched for HSCs, compared to only 6.9 (5.3-9.0)% with G-CSF (p&amp;lt;0.001, n=3 donors). Mechanistically, MGTA-145 bound to CXCR2 on neutrophils and led to a modest and transient increase in plasma concentrations of matrix metalloproteinase 9 (MMP- 9), a downstream target on neutrophils. To assess engraftment, we transplanted mobilized peripheral blood cells from healthy donors after a 5-day regimen of G-CSF or a single dose of plerixafor alone or MGTA-145 + plerixafor at limit dilution into sublethally irradiated primary and secondary NSG mouse recipients (n=3 cell doses, n=7-8 mice/group). Multilineage human engraftment was measured by flow cytometry 16 weeks post-transplant and SCID-repopulating cell (SRC) number was calculated (Figure 1A). MGTA-145 + plerixafor mobilized grafts (n=4 donors) led to a 23-fold increase in engraftment compared to G-CSF mobilized grafts (p&amp;lt;0.001, n=3 donors) and 11-fold higher engraftment compared to plerixafor mobilized grafts (p&amp;lt;0.001, n=3 donors). Immune cell subsets (B, T, and NK cells and cell subsets) mobilized by MGTA-145 + plerixafor were similar to those mobilized by plerixafor alone. While CD3+ T-cell numbers were comparable between MGTA-145 + plerixafor and plerixafor alone, MGTA-145 + plerixafor mobilized 0.2 (0.0-0.6) x108/kg CD8+ T-cells, constituting 1.8 (0.5-4.8)% of the graft, a number and proportion significantly lower than that mobilized by either G-CSF or plerixafor alone. To determine the effect of the mobilization regimen on xenogeneic GvHD, we developed a xenograft GvHD model in NSG mice where 6x106 PBMCs from various graft sources were infused into sublethally-irradiated animals (n=3-6 donors per graft source). Notably, MGTA-145 + plerixafor mobilized grafts resulted in significantly less GvHD than G-CSF (p&amp;lt;0.01) or plerixafor (p&amp;lt;0.001) grafts (Figure 1B). In vivo cellular subset depletion studies suggested that the GvHD protective effect in MGTA-145 + plerixafor grafts may be in part due to immunosuppressive monocytes which were not present, or present to a lesser degree, in grafts from donors mobilized with G-CSF or plerixafor. Conclusions . These data demonstrate that MGTA-145 + plerixafor is a rapid, reliable, and G-CSF free method to obtain high numbers of HSCs with durable engraftment potential and a graft with highly immunosuppressive properties. These data suggest that MGTA-145 + plerixafor is an effective single-day mobilization/collection regimen for both autologous and allogeneic stem cell transplantation resulting in enhanced engraftment and reduced GvHD in this xenograft model. Disclosures Goncalves: Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company, Patents &amp; Royalties. Hyzy:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hammond:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Falahee:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Howell:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Pinkas:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Schmelmer:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hoggatt:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company. Scadden:Magenta Therapeutics: Consultancy, Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Devine:Magenta Therapeutics: Consultancy. DiPersio:Magenta Therapeutics: Membership on an entity's Board of Directors or advisory committees. Savage:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company. Davis:Magenta Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Ex Vivo Expansion with E478 Increases the Rate of CRISPR-Mediated Homology Directed Repair of the Beta-Globin Gene in Human Hematopoietic Stem Cells By >10-Fold and Improves In Vivo Engraftment By >120-Fold

Background . Site-specific gene correction of hematopoietic stem cells (HSCs) via homology directed repair (HDR) has the potential to precisely repair defective genes and provide life-long cures for a variety of blood-based diseases. It is possible to obtain high levels of HDR during in vitro HSC culture, but these cells fail to robustly engraft in vivo, suggesting that the procedure of HDR compromises HSC function or that true HSCs are not undergoing HDR. Cells need to be actively cycling in order to undergo HDR, but conditions that allow HSC replication in vitro without compromising HSC number and function remain elusive. Thus, most HDR protocols minimize time in culture, potentially limiting HDR rates and cell yield. We recently reported that ex vivo expansion of HSCs with an aryl hydrocarbon receptor (AHR) antagonist is a clinically validated method to expand HSCs. The AHR antagonist-expanded CD34+ cell therapy, MGTA-456, results in rapid and durable recovery in patients with hematologic malignancies and inherited metabolic diseases (Wagner et al Cell Stem Cell 2016; Orchard et al AAN 2019). To apply this technology to gene-modified HSCs, we developed a novel AHR antagonist, E478, which expands NSG-engrafting cells 10-fold compared to uncultured primary human mobilized peripheral blood (mPB) CD34+cells in limit dilution studies. We previously showed that expansion with E478 results in up to 10-fold higher engraftment of lentiviral vector (LVV)-transduced cells and CRISPR/Cas9 knockout cells (Hoban et al ASGCT 2019). Here, we demonstrate that ex vivo expansion of mPB CD34+ cells with E478 results in &gt;10-fold increase in rate of HDR and &gt;120-fold increase in NSG engraftment of HDR+ cells compared to conventional approaches. Results . To determine whether more active cycling would lead to higher rates of HDR, we cultured cells for 1, 2, 3, and 4 days prior to electroporation with CRISPR gRNA targeting the beta-globin gene and transduction with a GFP-containing adeno-associated virus (AAV) donor template. Cell cycle analysis revealed that 33±1.8% of cells enriched for HSCs (CD34+CD90+ cells) remain quiescent after 2 days in culture, whereas 0.92±0.06% of CD34+CD90+ cells were quiescent after 3 and 4 days in culture (n=2 mPB donors). We then assessed HDR rates and HSC number after 1, 2, 3, and 4 days of additional culture. Compared to a conventional HDR protocol utilizing a 2-day pre-stimulation period followed by 1 day of culture after electroporation (herein called a 2+1 culture), we observed up to 8-fold increase in HDR with longer pre-stimulation periods, but this was accompanied with differentiation of CD34+CD90+ cells and loss of engraftment in NSG mice (79% decrease, p&lt;0.001). We next evaluated whether E478 could increase the dose of HSCs and maintain high HDR rates. We cultured mPB CD34+ cells with E478 for a 4 day pre-stimulation, performed HDR, and continued the expansion for 4 days with E478 (herein called 4+4 culture). With the 4+4 protocol, we observed a 6-fold increase in the rate of HDR in vitro and a 134-fold increase in the number of CD34+CD90+ cells with E478 relative to 2+1 conditions with DMSO vehicle (n=2, p&lt;0.01). Transplant of these cells into sublethally-irradiated NSG mice resulted in a 4-fold higher rate of engraftment (Figure A, p&lt;0.01, n=8 mice), 12-fold higher rates of HDR (Figure B, p&lt;0.001) and &gt;120-fold increase in the number of HDR+ NSG-engrafting cells relative to 2+1 cultures (Figure C, p&lt;0.001). Further, a 2+1 culture with E478 led to an 8-fold increase in number of HDR+ NSG-engrafting cells (p&lt;0.001) relative to standard 2+1 approaches without a small molecule. Multi-lineage engraftment was observed in all groups. Studies using E478 with bone marrow from patients with sickle cell disease are in progress and will be presented. Conclusions. We demonstrate that ex vivo HSC expansion with E478 enables higher rates of HDR and a high dose of HDR+ HSCs, leading to &gt;120-fold increase in the engraftment of HDR+ HSCs compared to conventional 2+1 approaches. Culture with E478 is a promising approach to realize the full potential of targeted gene correction in HSCs for a variety of genetic diseases. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. George:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents &amp; Royalties.

Nicotinamide (NAM) Modulates Transcriptional Signature of Ex Vivo Cultured UCB CD34+ Cells (Omidubicel) and Preserves Their Stemness and Engraftment Potential

Historical efforts at expansion of umbilical cord blood (UCB) derived CD34+ hematopoietic stem cells (HSCs) ex vivo with cytokines yielded large numbers of progenitors for transplantation but impaired their long-term engraftment ability. We used nicotinamide (NAM), an allosteric inhibitor of NAD-enzymes, to create omidubicel, an investigational cell therapy designed to improve the expansion of CD34+ HSCs for bone marrow transplant. A Phase 1/2 clinical study of omidubicel in patients with high-risk hematologic malignancies showed rapid neutrophil engraftment and a more favorable immune reconstitution profile in patients compared to historical controls.1 We hypothesized that NAM treatment maintains the stemness and engraftment potential of omidubicel, which is associated with clinical benefit.2 We performed transcriptome, transcription factor (TF), and pathway analysis by next generation sequencing (NGS) to discern the mechanism of action of NAM and to elucidate the pathways leading to the preservation of engraftment after ex vivo expansion of omidubicel compared to CD34+ cells grown in the absence of NAM. Transcriptome analysis revealed that treatment of CD34+ cells with cytokines alone (stem cell factor [SCF], thrombopoietin [TPO], IL-6, and FLT3 ligand) led to an increase in pathways responsible for cell proliferation and differentiation, apoptotic stress, and production of reactive oxygen species (ROS), and matrix metalloproteinases (MMPs), all of which were attenuated by NAM. TF enrichment analysis of NAM-upregulated genes and downregulated genes demonstrated that NAM modulated several TFs critically involved in pathways of HSC cell self-renewal, differentiation, apoptosis and migration. Specifically, NF-kB, C-Jun, LXR/RXR and PPARα/RXRα, and AMPK-mTor signaling were all reduced in NAM-treated CD34+ cells compared to controls. Reduced expression of key genes involved in the production of ROS and reactive nitrogen species (RNS) including NADPH-oxidase-related genes (CYBB, NCF2 and NCF4) and iNOS, suggested that NAM-expanded CD34+ cells were less exposed to oxygen and nitrogen free radical stress than controls. NAM also downregulated the expression of several matrix metalloproteinases (MMP) genes including MMP7, MMP9, MMP12 and MMP19. NAM-induced downregulation of MMPs may explain the increase in engraftment in patients receiving omidubicel. Pathway analysis of differentially expressed (DE) genes was conducted using ingenuity (IPA) software. IPA analysis of DE genes showed significant downregulation of growth factor activating pathways including SCF, TPO, FLT, and GM-CSF and Endothelin-1 and P2Y Purigenic Receptor, which was confirmed by a reduction in cell cycling rates of labeled cells. IPA analysis also pointed to genes in 3 key cellular pathways that were downregulated by NAM: stress induction of apoptosis, production of ROS and RNS, and production of MMPs. NAM treatment also uniquely upregulated genes linked to cellular metabolism including the Sirtuin family genes, TCA cycle genes, and HIF1a. Interestingly, NAM upregulated genes responsible for telomerase expression further validating our hypothesis that NAM preserves cell stemness. In summary, NGS transcriptome analysis revealed that ex vivo expansion of UCB derived CD34+ cells in the presence of NAM attenuated TFs responsible for proliferation and differentiation of stem cells. In addition, NAM treatment downregulated genes regulating the production ROS, RNS, and MMPs and upregulated genes controlling metabolism and senescence, thus allowing for the expansion of CD34+ cells with preserved function and long-term engraftment ability. Our gene expression data leads to a better understanding of the mechanisms by which NAM modulates CD34+ cells in omidubicel to preserve their function. These data provide further scientific rationale for the favorable clinical engraftment and patient outcomes observed in the Phase 1/2 clinical study of omidubicel.1 An international, randomized, multi-center Phase 3 study of omidubicel in patients with high-risk hematologic malignancies is underway.2 [1]Horwitz M.E., et. al., J Clin Oncol. 2019 Feb 10;37(5):367-374. [2] ClinicalTrials.gov identifier NCT02730299. Disclosures Lodie: Gamida Cell: Employment, Equity Ownership. Adams:Gamida Cell: Employment, Equity Ownership. Yackoubov:GAMIDA CELL: Employment, Other: unexecuted shares of the company . Peled:Gamida Cell: Employment, Equity Ownership.

MGTA-456, a First-in-Class Cell Therapy Produced from a Single Cord Blood Unit, Enables a Reduced Intensity Conditioning Regimen and Enhances Speed and Level of Human Microglia Engraftment in the Brains of NSG Mice

Background. Allogeneic bone marrow transplant (BMT) is a promising, curative approach for patients with inherited metabolic disorders (IMDs), a class of pediatric diseases characterized by a single enzyme deficiency. The goal of transplant is to provide cells that produce functional enzymes otherwise deficient in these patients, and thereby prevent or ameliorate neurological complications associated with selected IMDs. Donor-derived microglial cells are protective, limiting neurological disease progression. For IMD patients who do not have an HLA matched, non-carrier related donor, cord blood (CB) is the preferred HSPC source given its rapid availability and superior clinical outcomes compared to other graft sources. CB, however, is associated with delayed hematopoietic recovery and relatively poor engraftment due to the limited numbers of hematopoietic stem cells (HSCs) in a CB unit, delaying enzyme/protein reconstitution and cross-correction of non-hematopoietic cells.An aryl hydrocarbon receptor antagonist (AHRa)-based culture has been shown to expand CB CD34+ and CD34+CD90+ cells 330-fold and 100-fold, respectively, leading to rapid hematopoietic recovery after infusion of the clinical product, MGTA-456 (Wagner et al., Cell Stem Cell 2016 and Orchard et al., ASH 2018). As microglia are thought to be derived from HSCs, we hypothesized that MGTA-456 might lead to faster and greater microglial engraftment and potentially enable reduced intensity conditioning. Here, we assessed human hematopoietic and brain engraftment in NSG mice after transplant with MGTA-456 and showed that microglia engrafted faster with MGTA-456, less conditioning was needed, and that, mechanistically, these cells are derived from the CD34+CD90+ cell fraction.Methods. CB CD34+ cells were expanded in growth factor-supplemented media with or without an AHRa for 10 days. NSG mice were transplanted with unmanipulated CB CD34+ cells or the expanded product after 200 cGy total body irradiation or busulfan (BU) dosed at 20 or 40 mg/kg ip. Microglial engraftment was measured by flow cytometry of homogenized brains, quantitating the number of CD45+CD11b+Iba1+ cells, and by immunohistochemistry of brain sections.Results. Relative to naïve, unmanipulated CB CD34+ cells, transplant of MGTA-456 into sublethally irradiated mice led to an 8-fold increase in hematopoietic engraftment and a 10-fold increase in microglial engraftment in the brain (p<0.0001, n=15 mice), with histology consistent with engrafting microglia. As high dose BU enables enhanced microglia engraftment relative to irradiation by crossing the blood brain barrier and clearing host microglia (Capotondo et al., PNAS 2013), we evaluated the effectiveness of MGTA-456 after BU conditioning at 20 or 40 mg/kg. Transplant of MGTA-456 led to a 37-fold increase in engraftment relative to mice transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). Notably, transplant of MGTA-456 into mice conditioned with low-dose BU (20 mg/kg) led to a 15-fold increase in engraftment relative to high-dose BU (40 mg/kg)-conditioned animals transplanted with unmanipulated CB CD34+ cells (p<0.001, n=8). To evaluate speed of microglial engraftment, we evaluated brains weekly to 16 weeks after transplant. A 28-fold increase in microglial engraftment was demonstrated as early as 2 weeks post-transplant with MGTA-456 (p<0.0001, n=8). Number of engrafting hematopoietic cells in the periphery correlated with number of engrafting microglia in the brain (p<0.0001). Lastly, subpopulations of MGTA-456 were evaluated to determine the source of microglial engraftment. Only CD34+CD90+ cells, but not CD34+CD90- or CD34- cells, led to brain engraftment, consistent with the subpopulation of cells that result in hematopoietic engraftment following transplant of unexpanded cells (Radtke et al., Sci Trans Med 2017 and Goncalves et al., Blood 2017 130:659).Conclusions. These studies demonstrate that microglial engraftment is faster and greater in recipients of MGTA-456 even after lower dose BU conditioning, that microglial engraftment correlates with peripheral blood recovery, and that microglia cells are derived from CD34+CD90+ cells. These results suggest that lower dose BU may improve safety without jeopardizing efficacy in IMD recipients of MGTA-456. A Phase 2 clinical trial is ongoing to evaluate transplant of MGTA-456 in patients with select IMDs. [Display omitted] DisclosuresGoncalves:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Li:Magenta Therapeutics: Employment, Equity Ownership. Brooks:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

Oxidative stress preconditioning of mouse perivascular myogenic progenitors selects a subpopulation of cells with a distinct survival advantage in vitro and in vivo

Cell engraftment, survival and integration during transplantation procedures represent the crux of cell-based therapies. Thus, there have been many studies focused on improving cell viability upon implantation. We used severe oxidative stress to select for a mouse mesoangioblast subpopulation in vitro and found that this subpopulation retained self-renewal and myogenic differentiation capacities while notably enhancing cell survival, proliferation and migration relative to unselected cells. Additionally, this subpopulation of cells presented different resistance and recovery properties upon oxidative stress treatment, demonstrating select advantages over parental mesoangioblasts in our experimental analysis. Specifically, the cells were resistant to oxidative environments, demonstrating survival, continuous self-renewal and improved migration capability. The primary outcome of the selected cells was determined in in vivo experiments in which immunocompromised dystrophic mice were injected intramuscularly in the tibialis anterior with selected or non-selected mesoangioblasts. Resistant mesoangioblasts exhibited markedly enhanced survival and integration into the host skeletal muscle, accounting for a more than 70% increase in engraftment compared with that of the unselected mesoangioblast cell population and leading to remarkable muscle recovery. Thus, the positive effects of sorting on mesoangioblast cell behaviour in vitro and in vivo suggest that a selection step involving oxidative stress preconditioning may provide a novel methodology to select for resistant cells for use in regenerative tissue applications to prevent high mortality rates upon transplantation.

Engraftment of Human MDS Samples in Xenotransplanted Mice Depends on Human Cytokines but Not Mesenchymal Stem Cells (MSC)

Background: Myelodysplastic syndromes (MDS) are a heterogeneous group of diseases with limited treatment options. Development of a patient derived xenotransplantation model for pre-clinical studies is a priority goal in the field. It has been hypothesized that patient derived mesenchymal stem cells and/or human cytokines are necessary to establish MDS engraftment in immunocompromised mice. In this study we evaluated the ability of mesenchymal stem cells (MSC) to facilitate the engraftment process having as a goal to generate a robust patient-derived xenograft (PDX) mouse model for MDS. We also assessed the contribution of human cytokines as expressed in transgenic mice.Methods: MSC from normal donors and MDS patient samples were generated and characterized using standard culture methods. NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) and NOD.Cg-Prkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG)1Eav/MloySzJ (NSG-S) mice were used as recipients. Mice were injected with 1 x 106 bone marrow mononuclear cells (MNC) alone or in combination with 5 x 105 MSC. MSC were from normal donors, from same patient as MNC (autologous), from other MDS patients or normal donors (allogeneic). All cells were injected intrafemorally. Mice were assessed for engraftment by bone marrow aspiration at 6-8, 10 weeks and at necropsy at 12-32 weeks (depending on initial assessment). Long-term engrafted samples were further assessed for presence of stem cells by secondary transplantation into NSG-S mice. 1 x 106 human CD45+ cells were passaged without or with 5 x 105 MSC. Secondary recipients were assessed at 12 weeks for engrafment. MDS samples represented both high risk (refractory anemia with excess blasts, RAEB)) and low risk (refractory anemia (RA), refractory cytopenia with myelodysplasia (RCMD), chronic myelomonocytic leukemia (CMML)) disease. The overall degree of engraftment was assessed by bone marrow aspiration and measurement of human CD45+ cells by means of FACS. Lymphoid, myeloid and erythroid engraftment was assessed similarly. 5 x 105 MSCs from normal donors were transduced with GFP (pELNS.CBR-T2A-GFP) and intrafemorally injected into NSG mice and the presence of human MSC was evaluated by in vivo Imaging (Xenogen Spectrum system and Living Image Version 4.3 software.).Results: 12 out of 12 injected MDS samples showed persistent human cells when assessed at 6-8 weeks. For most samples, engraftment at that time-point was low (<2%) and was not consistently influenced by the presence or absence of MSC. On week 10, 4 out of 12 injected MDS patient samples showed increased engraftment and only one showed higher engraftment levels in the mice co-transplanted with MSC. Two of these were from patients with RAEB and CMML and showed both myeloid and erythroid engraftment. Molecular analysis showed consistent engraftment of the malignant clone in 2 out of 2 patient samples tested. Overall, only 3 out of 12 patients showed long-term engraftment (> 12 weeks) and these samples were transferred to secondary animals. Secondary transplanted mice injected with selected hCD45+ cells with or without MSC showed variable engraftment levels on week 10 after injections. One of them reached long-term engraftment (>12 weeks), whereas the second gradually lost engraftment after week 10 and the third is ongoing. MSC tracking showed a gradual loss of MSC after intrafemoral injections to the point that no MSC were detected 3 weeks later. Comparison of engraftment in NSG mice to NSG-S mice (the latter producing human cytokines) showed a slight increase in engraftment at week 6 which continued at later time points.Conclusions: Passive transfer of MDS hematopoietic cells as assessed at 6-8 weeks after intrafemoral injection is highly consistent and not dependent on MSC when total MNC fraction is used. The latter may be explained since MSC are lost within the first three weeks after injections. Direct comparison of NSG vs. NSG-S mice showed that the presence of hGM-CSF, hIL-3 and h-SCF in the NSG-S mice increased the engraftment levels. Successful long-term engraftment of MDS cells in xenotransplanted mice was achieved for one patient sample so far, indicating that MDS initiating cells can be maintained in NSG-S mice. However, establishing long-term engraftment can only be achieved with a subset of patient samples. MDS engraftment is influenced by human cytokines but not with MSC cells which may suggest that MDS does not depend on MSC's for long term growth. DisclosuresNunez-Cruz:Novartis: Research Funding. Milone:Novartis: Patents & Royalties, Research Funding.

CD93 Is a Novel Biomarker of Leukemia Stem Cells in Chronic Myeloid Leukemia

The introduction of BCR-ABL tyrosine kinase inhibitors (TKIs) has revolutionized the treatment of chronic myeloid leukemia (CML). However, although the majority of patients with chronic phase (CP)-CML obtain durable complete cytogenetic and major molecular responses, there is low level disease persistence postulated to be due to a population of TKI-insensitive leukemia stem cells (LSC). The aims of this study were (1) to fully characterize differences in gene expression between normal hematopoietic stem cells (HSC) and CP-CML LSC and (2) identify potential novel therapeutic targets specific to CML LSC.Lin-CD34+CD38- CD45RA-CD90+ normal HSC (n=3) and CP-CML LSC (n=6 patients at diagnosis), populations were isolated using a FACSAria and applied to Affymetrix HuGene 1.0ST arrays. The raw data (.CEL files) was imported into Partek Genomics Suite and Ingenuity Pathway Analysis software and principal component analysis and gene ontology ANOVA performed. A total of 1217 genes were significantly deregulated between normal HSC and CP-CML LSC. The most significantly deregulated genes and pathways were involved with the molecular and cellular functions of cell cycle, cell assembly and organisation, cellular movement, cell death and DNA replication, recombination and repair. These results suggested that CML LSC were less quiescent than normal HSC. Importantly, complimentary functional studies indicated that CML LSC have significantly increased proliferation (14 fold expansion; P<0.001) compared to normal HSC (no expansion) after 5 days in vitro culture. In addition, equivalent numbers of CML LSC produce ~4-fold more colonies in colony forming cell (CFC) assays than normal HSC (329±56 versus 86±17 per 2,000 cells, respectively; P<0.05). Fluorescence in situ hybridisation (FISH) demonstrated that >90% of lin- CD34+ CD38- CD45RA- CD90+ CML LSC from all patient samples were BCR-ABL positive (+).In addition to these deregulated intracellular pathways, we sought to assess if there were differences in expression of cell surface molecules that may be amenable to therapeutic manipulation. Of particular interest, our microarray studies demonstrated that CD93 was highly upregulated in CP-CML LSC (6 fold, p = 2.5x10-6). Increased CD93 expression was validated by Fluidigm digital PCR (6 fold increase, p = 0.02; n=6). Furthermore, using flow cytometry, we demonstrated significant upregulation of CD93 protein expression on lin-CD34+ CD38- CD45RA- CD90+ CML LSC from peripheral blood and bone marrow of CP-CML patients (n= 17; mean = 63.8% CD93+) compared to normal HSC from healthy peripheral blood stem cell donors (n=7; mean = 0.8% CD93+) and bone marrow donors (n=4; mean = 0.2% CD93+; p < 0.0001). FISH confirmed that 100% of lin-CD34+ CD38- CD90+ CD93+ CML cells were BCR-ABL+ in all samples assessed.CD93 (also known as C1qRp) is a C-type lectin-like domain (CTLD)-containing glycoprotein which regulates phagocytosis, with roles in cell adhesion and leukocyte migration. It is normally expressed on endothelial cells, hematopoietic precursors and mature cells including neutrophils, monocytes and platelets. Previous studies have shown CD93 to be upregulated in a proportion of AML patients (Saito et al, Sci Transl Med, 2010. 2(17): p. 17ra9).Short term (24h) in vitro exposure of lin-CD34+CD38- CD45RA- CD90+ CML LSC to TKIs (Imatinib or Dasatinib; n=3) reduced, but did not fully eliminate CD93 expression (Imatinib, 48.5% to 22.9%; Dasatinib, 47.7% to 9.2%). Importantly, following long-term TKI treatment of patients, lin-CD34+CD38-CD45RA-CD90+ cells from CP-CML patient bone marrow samples (n=2) taken in major molecular response demonstrated a small, but persistent population of CD93+ LSC which were BCR-ABL+ by FISH. Furthermore, in xenograft transplantation experiments (n = 5), after 16 weeks, CD34+CD93+ CML LSC engrafted lethally irradiated NOD/SCID/IL-2Rg-/- (NSG) mice with BCR-ABL+ cells, whereas CD34+CD93- cells from the same patient samples failed to engraft to significant levels (3.5-30 fold increase in engraftment with CD34+CD93- cells; p < 0.03). FISH confirmed that engrafted human cells were BCR-ABL+.Taken together, our results identify CD93 as a potential novel biomarker of CML LSC, which may also be helpful in assessing minimal residual disease at the LSC level. Further studies are ongoing to assess the therapeutic potential of inhibiting CD93 in CML LSC. DisclosuresCopland:Ariad: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

No Synergistic Effect of Cotransplantation of MSC and Ex Vivo TPO-Expanded CD34(+) Cord Blood Cells on Platelet Recovery and Bone Marrow Engraftment in NOD SCID Mice.

After cord blood (CB) transplantation, early platelet recovery in immune-deficient mice is obtained by expansion of CB CD34(+) cells with thrombopoietin (TPO) as single growth factor. Moreover, improvement of hematopoietic engraftment has been shown by cotransplantation of mesenchymal stem cells (MSC). We investigated whether a combination of both approaches would further enhance the outcome of CB transplantation in NOD SCID mice. NOD SCID mice were transplanted with either CB CD34(+) cells, CD34(+) cells with MSC, TPO-expanded CD34(+) cells or TPO-expanded CD34(+) cells with MSC. We analyzed human platelet recovery in the peripheral blood (PB) from day 4 after transplantation onward and human bone marrow (BM) engraftment at week 6. The different transplants were assessed in vitro for their migration capacity and expression of CXCR4. TPO expansion improved the early platelet recovery in the PB of the mice. Cotransplantation of MSC with CD34(+) cells improved BM engraftment and platelet levels in the PB 6 weeks after transplantation. Combining TPO expansion and MSC cotransplantation, however, neither resulted in a more efficient early platelet recovery, nor in a better BM engraftment, nor even very low or absent BM engraftment occurred. In vitro, MSC boosted the migration of CD34(+) cells, suggesting a possible mechanism for the increase in engraftment. Our results show that cotransplantation of MSC with TPO-expanded CD34(+) cells at most combines, but does not increase the separate advantages of these different strategies. A combination of both strategies even adds a risk of non engraftment.

Impact of islet size on pancreatic islet transplantation and potential interventions to improve outcome.

Better results have been recently reported in clinical pancreatic islet transplantation (ITX) due mostly to improved isolation techniques and immunosuppression; however, some limitations still exist. It is known that following transplantation, 30% to 60% of the islets are lost. In our study, we have investigated 1) the role of size as a factor affecting islet engraftment and 2) potential procedural manipulations to increase the number of smaller functional islets that can be transplanted. C57/BL10 mice were used as donors and recipients in a syngeneic islet transplant model. Isolated islets were divided by size (large, >300 μm; medium 150-300 μm; small, <150 μm). Each size was transplanted in chemically induced diabetic mice as full (600 IEQ), suboptimal (400 IEQ), and marginal mass (200 IEQ). Control animals received all size islets. Engraftment was defined as reversal of diabetes by day 7 posttransplantation. When the superiority of smaller islets was observed, strategies of overdigestion and fragmentation were adopted during islet isolation in the attempt to reduce islet size and improve engraftment. Smaller islets were significantly superior in engraftment compared to medium, large, and control (all sizes) groups. This was more evident when marginal mass data were compared. In all masses, success decreased as islet size increased. Once islets were engrafted, functionality was not affected by size. When larger islets were fragmented, a significant decrease in islet functionality was observed. On the contrary, if pancreata were slightly overdigested, although not as successful as small naive islets, an increase in engraftment was observed when compared to the control group. In conclusion, smaller islets are superior in engraftment following islet transplantation. Fragmentation has a deleterious effect on islet engraftment. Islet isolations can be performed by reducing islet size with slight overdigestion, and it can be safely adopted to improve clinical outcome.

Superselective intra-arterial umbilical cord blood administration to BM in experimental animals.

Umbilical cord blood (UCB) as a source of hematopoietic stem cells for transplantation is limited by the low number of cells and delayed engraftment. UCB cells are infused i.v. for transplantation, although only a proportion of the cells reach the BM. We investigated whether UCB could be administered safely using superselective intra-arterial (i.a.) injection. We injected human UCB (5 × 106) into the aorta in rats, into the iliac artery in mice and into the femoral nutrient artery (FNA) in rabbits. We used angiography, immunohistochemistry, intravital microscopy and qPCR to assess safety end points and the distribution of injected cells. All animals showed normal behavior. No evidence of organ infarction was noted. UCB injected into the FNA of rabbits did not change the flow rates, measured by angiography. By qPCR, we found significantly higher fold-change values in the injected BM compared with i.v. injection (P=0.0087). Using intravital microscopy we visualized the mouse capillary bed during i.a. injection without cellular congestion. In summary, we show that i.a. infusion of UCB is safe and reaches an eightfold increase in engraftment in the BM compared with i.v. infusion. These studies lay the foundation for clinical trials.

In utero depletion of fetal hematopoietic stem cells improves engraftment after neonatal transplantation in mice

AbstractAlthough 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.

Mesenchymal Stem Cell-Derived Vesicles Reverse Hematopoietic Radiation Damage

Extracellular vesicles, including exosomes and microvesicles, have been shown to epigenetically modulate cell fate (Aliotta et al JEV, 2013). We have evaluated the effect of murine or human mesenchymal stem cell (MSC)-derived vesicles on marrow from irradiated mice or on the marrow cell line, FDC-P1, irradiated in vitro. B6.SJL mice were exposed to 0 or 100 cGy whole body irradiation and 7 days later marrow was harvested, lineage depleted and then cultured with or without the addition of murine MSC-derived vesicles for 48 hours. The cells were then engrafted into 200 cGy exposed C57BL/6J mice and engraftment was analyzed at 3, 12, 24 and 36 weeks post-transplant. Vesicle exposure led to a statistically significant increase in engraftment by the irradiated cells at 24 and 36 weeks with the average percent donor engraftment equal to 8.8 ± 4% and 9.6 ± 5% for the vesicle treated group compared to 3.7 ± 2% and 3 ± 2% for the non-vesicle treated groups at 24 weeks and 36 weeks, respectively (Figure 1). In a similar experiment, murine marrow was harvested 24 hours after 100 cGy whole body irradiation, and cultured with and without vesicles for 24 hours. There was a statistically significant increase in donor engraftment by the Lineage negative cells incubated with MSC-derived vesicles at 3 and 6 months (Figure 2). The increase in engraftment potential with vesicle exposure persisted in serial transplantation. These studies indicate that MSC-derived vesicles can reverse radiation damage to marrow when administered either 1 or 7 days after irradiation. The in vivo effect of murine MSC-derived vesicles on post-radiation blood cell count recovery was tested. C57BL/6 mice were exposed to 500 cGy total body irradiation. MSC-derived vesicles were then injected intravenously 6, 24 and 72 hours after irradiation. WBC recovery compared to non- irradiated mice was 17% in the non-vesicle group and 37% in the vesicle- injected group at 14 days; the values at 28 days were 61 and 89%, respectively, indicating that vesicle injection enhanced leukocyte recovery following radiation damage. Marrow cells from the non-irradiated, the irradiated and the irradiated plus vesicle-treated mice were then analyzed for gene expression. Twelve genes were elevated in the irradiated cells and the elevations were reversed by vesicle exposure while 17 genes were depressed in the irradiated cells and the depressions reversed by vesicle exposure. Studies with the marrow cell line, FDC-P1, showed that apoptosis in irradiated cells was reversed by exposure to MSC-derived vesicles and growth of irradiated cells was restored. Restoration of growth by in vitro exposure to MSC-derived vesicles is shown in Figure 3. In summary, these data indicate that MSC-derived vesicles have the capacity to ameliorate radiation-induced damage to marrow and may represent an important biologic for use in radiation exposures. [Display omitted] [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

A Cell-Autonomous Myeloproliferative Phenotype Caused by Loss of Latexin in Stem and Progenitor Cells.

Abstract 2314We have previously shown a novel role for Latexin (Lxn), a carboxypeptidase inhibitor, in influencing the size of murine hematopoietic stem cell (HSC) population. Lxn negatively regulates HSC population size by enhancing apoptosis while repressing proliferation and self-renewal. However, the major mechanisms underlying this phenotype remain unknown. To further study the functionality of latexin in hematopoiesis, we generated a knock-out mouse model (Lxn−/− mouse) with a truncation of Lxn exon 2 to exon 5.Blood cell counts showed that lymphocytes, neutrophils, eosinophils, and monocytes were significantly increased from 50% to 100%, indicating an overall myeloid expansion in the periphery. The Lxn−/− mice also showed hypercellular bone marrow (BM), splenomegaly, and increased colony formation in methylcellulose.We found that the frequencies and absolute numbers of Lineage-/Sca-1+/c-Kit+ (LSK) and multi-potent progenitor (MPP) cells were significantly increased, while short-term and long-term HSCs remained unchanged in Lxn−/− mice. We next evaluated both lymphoid and myeloid progenitor subpopulations within the MPP compartment. Absolute numbers of granulocyte-monocyte progenitor (GMP) cells as well as common lymphoid progenitor (CLP) cells were significantly increased by 60–80% in Lxn−/− mice compared with wild type animals.We then performed cobblestone-area-forming-cell (CAFC) assay, an in vitro functional assay to evaluate the numbers of functional progenitors and stem cells. As a result, cobblestone area forming cells were significantly up-regulated at days 7, 14, 28 and 35, indicating that numbers of both mature progenitors and functional stem cells in BM were increased.To further investigate the observed progenitor and stem cell expansion in Lxn−/− mice, we studied the apoptotic status of BM cells, using the apoptotic marker Annexin V. The apoptosis frequencies were reduced approximately 50% in Lxn deficient LSK cells and MPP cells, suggesting that Lxn positively regulates apoptosis apoptosis in HSCs, as reported previously.Moreover, we used the competitive repopulation assay to determine functional capacities of Lxn−/− HSCs. In this assay, equal numbers (1×106) of Lxn−/− (or wild type) whole BM cells were transplanted along with competitors into lethally irradiated recipient animals. We analyzed the peripheral blood and BM of recipients at 15 weeks post transplantation. In both BM and PB, Lxn−/− cells showed a 50% increase in engraftment. We noted that there was significant up-regulation in granulocytes and monocytes (50%), as well as B cell lymphocytes (50%), but not in T cells, which is consistent with what we observed in Lxn−/− mice. These findings reveal that the myeloproliferation in Lxn−/− mice is transplantable and thus is intrinsic.To gain insights into the molecular mechanisms underlying the phenotypes created by loss of Lxn expression, we carried out gene profiling between Lxn and wild type MPP cells. Microarray experiments were performed on BM cells using Affymetrix Gene Chip. To study the pathway changes in response to loss of Lxn expression, we performed the gene set enrichment analysis (GSEA). Five enriched pathways have been identified as altered in Lxn−/− MPP cells that include: cell communication, ECM receptor interaction, proteasome, cell cycle and neuroactive ligand receptor interaction.In conclusion, these data showed that loss of Lxn leads to proliferative hematopoiesis with clonal expansion at the stem and progenitor cell levels, and that this myeloproliferative phenotype is cell autonomous and transplantable.This work was supported by NIH HD-060915 and the Edward P. Evans Foundation Disclosures:No relevant conflicts of interest to declare.

Improved Engraftment of Human Hematopoietic Stem Cells in NOD/SCID Mice by the Antioxidant, N-Acetyl-L-Cysteine

Abstract 1884Non-obese diabetic/severe combined immune-deficient (NOD/SCID) mice are widely used as a human-mouse xeno-transplant model for assessing engraftment of human hematopoietic stem cells (HSCs). Optimizing this model and understanding the variables that affect engraftment are critical to correctly interpret the engraftment results of human HSCs in NOD/SCID recipients. However, the engraftment efficiency of human HSCs remains very low in NOD/SCID mice. Larger efforts have been devoted to improving engraftment by increasing the immune-deficiency of the mice, but less effort has been made toward other negative parameters in the host. Our preliminary study showed that NOD/SCID mice had higher levels of reactive oxygen species (ROS) in the bone marrow (BM) in comparison with other commonly used mouse strains (C57BL/6 and BALB/C). Given the previous studies by us and others showing that excessive ROS could impair the function of HSCs and that antioxidants were able to overcome the exhaustion of mouse HSCs in transplant recipients, we hypothesized that the poor engraftment of human hematopoietic cells in NOD/SCID recipients may be partially attributed to higher levels of ROS in NOD/SCID BM and a reduction of ROS by antioxidants may improve the engraftment of human HSCs in NOD/SCID mice.To test this hypothesis, NOD/SCID (8- to 12-week-old) mice were injected subcutaneously daily with an antioxidant, N-acetyl-L-cysteine (NAC) or PBS (control) for two weeks before being irradiated with 200 cGy from a cesium-137 source at 70cGy/min. Different doses of CD34+ cells or highly-enriched HSCs from human cord blood (CB) were injected through the tail vein or into the right tibia of the mice. The mice were maintained on NAC treated drinking water following injection, then sacrificed 12–14 weeks after transplantation to measure the engraftment levels of different hematopoietic cell lineages.We found that treatment with NAC was able to lower the levels of ROS in NOD/SCID BM. At the highest dose of injected CD34+ cells (>5×105), the NAC treated recipients displayed a significant increase of engraftment (2.1-fold) when compared with the control group (Control vs. NAC treated recipients: 11.04±3.11% vs. 23.21±4.0%, p=0.0224; n=20/each). This improvement was even more significant when injected cell numbers were reduced (2×105 and 1×105 had 3.9- and 4.9- fold higher engraftment, respectively, in NAC treated recipients), thus suggesting that saturating levels of HSCs may ease the anti-oxidant effect on engraftment. Furthermore, we also demonstrated higher levels of overall engraftment and multi-lineage differentiation of human HSCs (Lin-CD34+CD38-CD45RA-CD90+CD49f+Rholow) with a limiting dilution analysis. In comparison with the control mice, NAC treated recipients displayed 2.5-, 3.5-, and 5.7-fold increases in engraftment in the injected tibia (IT), BM and spleen, respectively. The frequency of SCID-repopulating cell (SRC) in IT was approximately 3.0-fold higher in NAC treated mice than in control mice (1 in 108 vs. 1 in 36). Similar improvements (4.1- to 7.9- fold) in SRC frequencies were also detected in BM and spleen. Notably, NAC increased the probability of positive engraftment when a single human HSC was directly transplanted into the BM of NOD/SCID mice. In summary, our current study uncovers a previously unappreciated negative effect of ROS in the human-NOD/SCID xenotransplant model and reduction of ROS via antioxidants such as NAC may significantly enhance the engraftment of human hematopoietic stem cells in NOD/SCID mice. Disclosures:No relevant conflicts of interest to declare.

Loss of De Novo DNA Methylation Causes Expansion of the Mouse Hematopoietic Stem Cell Pool

AbstractAbstract 835DNA methylation is one of the major epigenetic modifications in the vertebrate genome and is catalyzed by the DNA methyltransferase enzymes Dnmt1, Dnmt3a and Dnmt3b. We observed a dynamic expression profile of Dnmt3a and Dnmt3b in the hematopoietic system with both enzymes expressed at exponentially higher levels in hematopoietic stem cells (HSCs) compared to progenitors and differentiated cells and hypothesized that some of the unique characteristics of HSCs were epigenetically regulated by Dnmt3a and Dnmt3b. To study this, we crossed Dnmt3a and -3b conditional knock-out (KO) mice to Mx1-cre mice to generate inducible single- and double-KO (dKO) mice. We performed competitive transplantation of HSCs (side-population+c-Kit+Lineage-Sca-1+ = SPKLS) from these mice along with wild-type whole bone marrow competitor and induced deletion of the Dnmt3's in the donor cells by sequential pIpC injections in the wild-type recipients. No dramatic differences were observed in primary recipients in the absence of other hematopoietic perturbation, however when we re-transplanted Dnmt3a- and Dnmt3b-KO HSCs into secondary recipients, they exhibited surprisingly high peripheral blood reconstitution compared to control HSCs (>4-fold increase in engraftment). This was reflected in the bone marrow of these mice with a corresponding >4-fold expansion of the HSC pool (phenotypically defined by any of SPKLS; CD34-Flk2-KLS; CD150+CD48-KLS) with virtually all of these cells being derived from the Dnmt3a- and Dnmt3b-KO donor HSCs. Consistent with a previous study, we observed a decline in functional output of Dnmt3a/3b-dKO HSCs in secondary transplants in terms of peripheral blood chimerism, but surprisingly these mice also exhibited a modest expansion of the HSC pool (∼2-fold), the majority of which were derived from donor Dnmt3a/3b-dKO HSCs. In subsequent tertiary and quaternary transplantation, Dnmt3 single-KO HSCs remained highly superior in peripheral blood engraftment capacity relative to control HSCs and Dnmt3a/3b-dKO HSCs (Figure 1), although the expansion of the HSC pool in all Dnmt3-KOs continued to varying degrees. This enhanced HSC activity appears to be a cell autonomous mechanism as purified Dnmt3-KO SPKLS cells from transplanted mice have much greater hematopoietic colony forming potential in vitro compared to control HSCs on a per cell basis. However the observed HSC expansion does not appear attributable to either enhanced proliferation of Dnmt3-KO HSCs or more resistance to apoptosis. The serially-transplanted Dnmt3-KO HSCs are not overtly transformed, in that the levels of differentiated blood cells are still normal and the mice appear to be healthy. This may be akin to a pre-malignant state seen in human myelodysplastic syndrome. We have performed microarray expression profiling of serially-transplanted Dnmt3-KO HSCs and identified several candidate genes which are currently being investigated as the mechanism for HSC expansion. Our data suggest ablation of de novo DNA methylation in HSCs uncouples normal self-renewal and differentiation. These studies present further evidence for the contribution of epigenetic regulation to stem cell activity and provide a tantalizing link between potential aberrant methylation in HSCs contributing to leukemic transformation. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

CD3+ T-Cell Content of Short Term Ex Vivo Expanded UCB on a MSC Platform Predicts Human Engraftment in a NOD/SCID Model.

Background. Umbilical cord blood (UCB), a source of hematopoeitic stem cells (HSC), is marked by delayed engraftment attributed to the limited cellular content of a single UCB unit. Cytokine-based ex vivo expansion of UCB is a way of increasing the number of cells available for allogeneic transplants, however, this strategy has not demonstrated improved engraftment in human clinical trials. Further studies have incorporated human mesenchymal stem cells (huMSC) which may provide signals that control the proliferation, survival, and differentiation of HSC. In attempt to reduce the occurrence and severity of GVHD following allogeneic transplants, strategies such as utilizing T-cell depleted grafts have been pursued, however, clinical trials using these grafts have shown decreased rates of donor engraftment, suggesting the requirement of accessory cells as well as HSC to achieve engraftment.Methods. UCB mononuclear cells (MNC) were cultured using cytokines (IL-3, IL-6, G-CSF, SCF, Flt-3L, EPO) with or without a feeder-layer of huMSC for 12 days. On day 12, viability, 4-color flow cytometry, and human engraftment potential were measured. Human engraftment potential was determined by injecting cells (without CD34+ selection) from each culture condition and non-cultured UCB MNC, via tail vein, into sublethal irradiated NOD/SCID mice. Mice were injected with unexpanded UCB MNC (n=23), UCB expanded in huMSC+cytokines (n=21) and UCB expanded in cytokines alone (n=10). 7–9 weeks following injection of human cells, bone marrow was harvested and analyzed for human content. Positive human engraftment was determined by a human %CD45+ of ≥ 0.4%.Results. An 8.77 fold expansion of UCB cultured in cytokines alone compared to a 7.14 fold expansion of UCB cultured in huMSC + cytokines was observed. Surface phenotyping of expanded UCB, and human cells emerging in the bone marrow of NOD/SCID mice following injection of cultured and non-cultured UCB are inTable 1UnexpandedhuMSC+cytokinesCytokines% CD344.0 (4.40M)2.04 (10.1M)1.52 (4.35M)% CD5617.0 (1.70M)7.07 (36.1M)3.69 (13.8M)% CD343.41 (.341M)2.25 (10.9M)3.52 (12.3M)Bone marrow of NOD/SCID mice% CD45+2.573.582.38% of CD45+ co-expressing CD311.09.2518.7% of CD45+ co-expressing CD1933.516.619.4% of CD45+ co-expressing CD5610.18.041.60Human engraftment was seen in 13 mice which received unexpanded UCB, 10 mice which received UCB expanded in huMSC+cytokines and only 3 mice which received UCB expanded in cytokines alone. Statistical analysis, using multivariable logistic regression to determine the factors that predict engraftment, revealed that the proportions of T and NK cells present in expanded UCB correlated with engraftment. A 10% increase in the proportion of CD45+ co-expressing CD3 was associated with a 1.79 fold increase in engraftment (p=0.016), whereas each 10% increase in the proportion of CD45+ co-expressing CD56 increased the odds of engrafting by 104% (p= 0.003).Conclusions. We observed an expansion of CD34 hematopoietic progenitors as well as a greater proportion of CD3+ cells, in expansion conditions incorporating huMSC. Additionally, we observed improved rates of engraftment in this expansion condition. Therefore, although the mechanism by which accessory cells including T and NK cells facilitate HSC engraftment is not known, we observed that the presence of accessory cells in addition to CD34 hematopoietic progenitors facilitated engraftment in NOD/SCID mice.