ALBERT

Description: Plerixafor (AMD3100) and granulocyte colony-stimulating factor (G-CSF) mobilize peripheral blood stem cells by different mechanisms. A rhesus macaque model was used to compare plerixafor and G-CSF–mobilized CD34+ cells. Three peripheral blood stem cell concentrates were collected from 3 macaques treated with G-CSF, plerixafor, or plerixafor plus G-CSF. CD34+ cells were isolated by immunoselection and were analyzed by global gene and microRNA (miR) expression microarrays. Unsupervised hierarchical clustering of the gene expression data separated the CD34+ cells into 3 groups based on mobilization regimen. Plerixafor-mobilized cells were enriched for B cells, T cells, and mast cell genes, and G-CSF–mobilized cells were enriched for neutrophils and mononuclear phagocyte genes. Genes up-regulated in plerixafor plus G-CSF–mobilized CD34+ cells included many that were not up-regulated by either agent alone. Two hematopoietic progenitor cell miR, miR-10 and miR-126, and a dendritic cell miR, miR-155, were up-regulated in G-CSF–mobilized CD34+ cells. A pre-B-cell acute lymphocytic leukemia miR, miR-143-3p, and a T-cell miR, miR-143-5p, were up-regulated in plerixafor plus G-CSF–mobilized cells. The composition of CD34+ cells is dependent on the mobilization protocol. Plerixafor-mobilized CD34+ cells include more B-, T-, and mast cell precursors, whereas G-CSF–mobilized cells have more neutrophil and mononuclear phagocyte precursors.

Description: Abstract 36 Bone marrow stromal cells (BMSCs, also known as “mesenchymal stem cells) are being used to treat acute graft-versus-host-disease, but their mechanisms of immune modulation are not certain. In vitro studies suggest that the immunosuppressive activity of BMSCs involves multiple factors including transforming growth factor β (TGF-β), hepatocyte growth factor (HGF), TNF-α, IFN-ψ, IL-10, IL-2 and prostaglandin E2 (PGE2). In this study we compared BMSCs with other types of stem cells using global transcriptome and microRNA (miR) expression analysis to identify factors that might contribute to their immunosuppressive effects and to identify biomarkers for assessing the stability, consistency, comparability, and potency of clinical BMSC products. BMSCs (passage 2 or 3) made from marrow aspirates of 4 healthy subjects by culturing in flasks and cell factories with 20% fetal bovine serum (FBS) were compared to 3 human embryonic stem cell lines (hES) and CD34+ cells isolated from G-CSF-mobilized peripheral blood from 3 healthy subjects. The cells were analyzed with an miR expression array with more than 800 probes and an oligonucleotide expression microarray with more than 35,000 probes. Hierarchical clustering analysis of the miR expression data separated the 3 types of cells into 3 distinct groups with unique signatures. MiRNA implicated in cancer and stem cell development that were up-regulated in BMSC compared to CD34+ cells and hES included miR21 and 125b and cancer and stem cell miR down-regulated in BMSCs included miR106a, 106b, 18a, 19b and 20b. When compared to CD34+ cells, several miR in the onco-miR17-92 cluster (miR17, 18a, 20a, 19b-1, and 92-1) and onco-miR106a-363 cluster (106a, 18b, 20b, 19b-2, 92-2, and 363) were down-regulated in BMSCs. Hierarchical analysis of the 4,600 genes that were expressed in greater than 80% of samples and were increased more than 2-fold in at least one sample clustered the 3 cell types into separate groups. Ingenuity pathway analysis revealed that the following pathways contained a significant number of genes that were up-regulated in BMSCs compared to both CD34+ cells and hES: actin based motility by rho, actin cytoskeletal signaling, integrin signaling, androgen signaling, IL-8 signaling VEGF signaling, PTEN signaling, oncostatin M signaling, fMLP signaling in neutrophils, inositol metabolism, cavelolar-mediated endocytosis and NRF2-mediated oxidative stress response pathway. DNA methylation and transcription repression pathway genes were down-regulated in BMSCs. Analysis of specific differentially expressed genes found that the gene most up-regulated in BMSCs was TGF-β1. Its expression was 346-fold fold greater in BMSCs than CD34+ cells and 298-fold greater than in hES. In addition, when compared to CD34+ cells, the expression of IL-6 was up-regulated 13.6-fold in BMSCs, prostaglandin E synthase was up-regulated 13.2-fold, and HGF 8.43-fold. These results support a possible role for TGF-β1, IL-6, HGF and PGE2 in BMSC-mediated immune modulation. TGF-β1, IL-6, HGF and PGE2 synthase are potential BMSC potency biomarkers, but further studies, including the correlation of the expression of these biomarkers in specific BMSC products with the clinical outcomes of patients treated with these products, are needed. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2523 Poster Board II-500 Prostaglandin E2 (PGE2), a member of the eicosanoid family, is a local signaling molecule released from mammalian blood vessel walls and other hematopoietic niches. PGE2 produces tissue specific effects by interacting with G protein receptors in cell membranes. It has been found to be an important regulator of hematopoietic stem cell (HSC) number during embryogenesis and is required for the development of HSCs (North et al., Nature 447:1007–1011, 2007). Additionally, PGE2 enhances HSC engraftment in competitive transplantation assays (North et al., Nature 447:1007–1011, 2007) through alterations in the survival, proliferation and homing of transplanted cells (Hoggatt et al., Blood 113:5444–5455, 2009). Immunoselected CD34+ cells were isolated from the leukapheresis products of both human and non-human primates following cytokine mobilization with G-CSF. CD34+ cells were incubated in X-Vivo 10™ with either 50μM or no PGE2 for one hour at 37°C on RetroNectin™ coated plates. After one hour incubation, media was replaced with X-Vivo 10™ supplemented with 10ng/mL SCF and IL-6 and cells were kept at 37°C and collected either prior to or at 2, 6, 12 and 24 hours following PGE2 exposure. Total RNA was isolated from cells and amplified to cRNA. Control cRNA was labeled with Cy3 dye and experimental cRNA was labeled with Cy5 dye. Control and experimental labeled cRNA was co-hybridized to a custom-made 17.5K cDNA (UniGene cluster) microarray. The relationship between the cells was analyzed using an unsupervised Eisen's hierarchical clustering method and gene regulation was analyzed at each time point using an Array scatter plot comparison. Statistical analysis was done using Array Class Comparison analysis and pathway analysis was carried out using Ingenuity Pathway Analysis. Many of the significantly up regulated genes were associated with pathways activated by PGE2, including genes involved in the inflammatory response, signal transduction, and G proteins. Interestingly the human CD34+ cells showed at 2 hours a 21.8-fold increase of 3′,5′-cyclic AMP phosphodiesterase mRNA consistent with prior observations in zebrafish and murine cells (Goessling et al. Cell 136: 1136–1148). Pathways that increased at 2 hours include molecular mechanisms of cancer, and those that were down regulated include CCR5, CTLA4, and leukocyte extravasation signaling. At 6 hours the molecular mechanisms of cancer pathway predominated even more (diminishing by 12 hours) while oxidative phosphorylation was down regulated. Genes involved in cAMP and WNT signaling are observed to be up regulated at earlier time points but diminish with time. These results correlate with protein expression observations that have been made in the zebrafish and murine models following PGE2 treatment (Goessling et al. Cell 136: 1136–1148) and are consistent with the observation that PGE2 may influence the self renewal of HSCs. Disclosures: Goessling: Fate Therapeutics: Consultancy, Patents & Royalties.

Description: G-CSF (Filgrastim) has been the standard agent for mobilizing peripheral blood stem cells (PBSCs) for transplantation. An alternative agent, AMD3100 (Plerixafor), has been used to mobilize PBSCs for autologous transplantation and is being tested in allogeneic donors. Since these agents have different mechanisms of action, the stem cells they mobilize may differ. We used a rhesus macaque model to compare CD34+ cells mobilized with these agents. Three macaques were given G-CSF, G-CSF plus SCF, AMD3100, or AMD3100 plus G-CSF. PBSCs were collected by apheresis and CD34+ cells with isolated by immunoselection. For G-CSF mobilization 10 μg/kg/day SQ was given for5 days, for G-CSF plus SCF mobilization 10 μg/kg/day of G-CSF and 200 μg/kg/day SQ of SCF were given for 5 days. For AMD3100 mobilization 1 mg/kg dose was given and for AMD3100 plus G-CSF mobilization 5 days of G-CSF (10 μg/kg/day) and one dose ofAMD3100 (1 mg/kg) were given. A PBSC concentrate was collected 2 hours after the dose of AMD3100 or the last dose of G-CSF. The CD34+ cells were analyzed by global gene and micro RNA (miR) expression analysis. A mean(SD) of 2.0(0.5)×107, 3.2(2) ×107, 2(0.5) ×107, and 12(7) ×107 immunoselected CD34+ were collected for G-CSF alone, G-CSF+SCF, AMD3100, and AMD3100+G-CSF, respectively. Gene expression analysis was performed with a microarray with 17,000 cDNA probes and miR analysis with an array with 827 miRs. Unsupervised hierarchical clustering of the gene expression data separated the samples into 2 clusters. One cluster included all CD34+ cells mobilized with G-CSF and G-CSF+SCF and the other all CD34+ cells mobilized with AMD3100and AMD3100+G-CSF. We found that 1,097 genes were differently expressed among the CD34+ cells mobilized with the 4 protocols (F-test, p