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Background Epigenetic modifications likely control fate of hematopoietic stem cells (HSC). The chromatin modifying agents (CMA), 5-aza-2’-deoxyctidine (5azaD) and trichostatin A (TSA) have previously been shown to expand HSC from cord blood and bone marrow. Here we assessed whether CMA can also expand HSCs present in growth factor mobilized human peripheral blood (MPB). Study Design & Methods 5azaD and TSA were sequentially added to CD34+ MPB cells in the presence of cytokines and the cells were cultured for nine days. Results Following culture, a 3.6 ± 0.5 fold expansion of CD34+CD90+ cells, a 10.1 ± 0.5 fold expansion of primitive colony forming unit (CFU)-mix, and a 2.2 ± 0.5 fold expansion of long-term cobble stone-area forming cells (CAFC) was observed in 5azaD/TSA expanded cells. By contrast, cells cultured in cytokines without 5azaD/TSA displayed no expansion; rather a reduction in CD34+CD90+ cells (0.7 ± 0.1 fold) and CAFCs (0.3 ± 0.1) from their initial numbers was observed. Global hypomethylation corresponding with increased transcript levels of several genes implicated in HSC self-renewal, including HOXB4, GATA2, and EZH2, was observed in 5azaD/TSA expanded MPB cells in contrast to controls. 5azaD/TSA expanded MPB cells retained in vivo hematopoietic engraftment capacity. Conclusion MPB CD34+ cells from donors can be expanded using 5azaD/TSA and these expanded cells retain in vivo hematopoietic reconstitution capacity. This strategy may prove to be potentially useful to augment HSCs numbers for patients who fail to mobilize.

394Neurofibromatosis type 2 (NF2) is an autosomal dominant disorder, characterized by the development of bilateral vestibular and spinal schwannomas, meningiomas, and ependymomas. 1,2 The NF2 gene encodes a 595 amino acid polypeptide known as NF2 protein or Merlin or Schwanomin. The primary structure of NF2 protein is homologous to the ERM family of peripheral membrane proteins, which includes Ezrin, Radixin, and Moesin. 3,4 The founding member of the ERM superfamily is the erythrocyte membrane protein 4.1, which cross-link's spectrinactin complexes and attaches them to the plasma membrane. 5 ABSTRACT: Background: Neurofibromatosis type 2 (NF2) is an autosomal dominant disease predisposing individuals to the risk of developing tumors of cranial and spinal nerves. The NF2 tumor suppressor protein, known as Merlin/Schwanomin, is a member of the protein 4.1 superfamily that function as links between the cytoskeleton and the plasma membrane. Methods: Upon selective extraction of membrane-associated proteins from erythrocyte plasma membrane (ghosts) using low ionic strength solution, the bulk of NF2 protein remains associated with the spectrin-actin depleted inside-out-vesicles. Western blot analysis showed a ~70 kDa polypeptide in the erythrocyte plasma membrane. Furthermore, quantitative removal of NF2 protein from the inside-out-vesicles was achieved using 1.0 M potassium iodide, a treatment known to remove tightly-bound peripheral membrane proteins. Results: These results suggest a novel mode of NF2 protein association with the erythrocyte membrane that is distinct from the known membrane interactions of protein 4.1. Based on these biochemical properties, several purification strategies were devised to isolate native NF2 protein from human erythrocyte ghosts. Using purified and recombinant NF2 protein as internal standards, we quantified approximately ~41-65,000 molecules of NF2 protein per erythrocyte. Conclusion: We provide evidence for the presence of NF2 protein in the human erythrocyte membrane. The identification of NF2 protein in the human erythrocyte membrane will make it feasible to discover novel interactions of NF2 protein utilizing powerful techniques of erythrocyte biochemistry and genetics in mammalian cells. RÉSUMÉ: Purification de la protéine codée par le gène suppresseur de tumeur NF2 à partir d'érythrocytes humains. Contexte : La neurofibromatose de type 2 (NF2) est une maladie dominante autosomique qui prédispose au développement de tumeurs au niveau des nerfs crâniens et des nerfs spinaux. La protéine codée par le gène suppresseur de tumeurs NF2, connue sous le nom de Merlin/Schwannomin, fait partie de la superfamille des protéines 4,1 impliquées dans l'interface entre le cytosquelette et la membrane plasmatique. Méthodes : Lors de l'extraction sélective des protéines associées à la membrane cellulaire d'érythrocytes plasmatiques (fantômes d'hématies) au moyen d'une solution dont la force ionique est faible, la majeure partie de la protéine NF2 demeure associée aux vésicules inversées dépourvu...