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HSCs are rare cells that have the unique ability to self-renew and differentiate into cells of all hematopoietic lineages. The lack of donors and current inability to rapidly and efficiently expand HSCs are roadblocks in the development of successful cell therapies. Thus, the challenge of ex vivo human HSC expansion remains a fertile and critically important area of investigation. Here, we show that either SALL4A- or SALL4B-transduced human HSCs obtained from the mobilized peripheral blood are capable of rapid and efficient expansion ex vivo by >10 000-fold for both CD34+/CD38− and CD34+/CD38+ cells in the presence of appropriate cytokines. We found that these cells retained hematopoietic precursor cell immunophenotypes and morphology as well as normal in vitro or vivo potential for differentiation. The SALL4-mediated expansion was associated with enhanced stem cell engraftment and long-term repopulation capacity in vivo. Also, we demonstrated that constitutive expression of SALL4 inhibited granulocytic differentiation and permitted expansion of undifferentiated cells in 32D myeloid progenitors. Furthermore, a TAT-SALL4B fusion rapidly expanded CD34+ cells, and it is thus feasible to translate this study into the clinical setting. Our findings provide a new avenue for investigating mechanisms of stem cell self-renewal and achieving clinically significant expansion of human HSCs.

[Figure][1] A 96-year-old woman with a past medical history of hypertension, medication noncompliance, and lack of medical follow-up was brought to the emergency room with altered mental status. She was to found to have malignant hypertension with concomitant acute pontine hemorrhage. Her

Annexin A5 (A5) is a potent anticoagulant protein that crystallizes over phospholipid surfaces, shielding them from availability for coagulation enzyme reactions. The antiphospholipid antibody (aPL) syndrome (APS) is an autoimmune thrombophilia that is marked by the presence of antibodies against phospholipid-binding proteins and the paradoxical lupus anticoagulant phenomenon. aPL antibodies can promote coagulation by interfering with the crystallization of A5, thereby increasing the exposure of blood to thrombogenic anionic phospholipids. Since a flow cytometric assay for aPL measuring A5 binding to frozen thawed platelets was previously reported, we investigated whether phosphatidylserine-bound polystyrene beads could provide a robust platform for the assay. Beads were incubated with sera from patients with the aPL syndrome and from non-aPL controls. Fluorescence-tagged A5 was added and samples were analyzed by flow cytometry. Similarly treated beads were also used in coagulation assays to determine whether A5 anticoagulant function was also inhibited. Control sera permitted fluorescent labeling of nearly all beads. In contrast, sera from aPL syndrome patients produced a major population of unlabeled beads, sometimes accompanied by a minor population of labeled beads. 7 of 13 confirmed aPL syndrome patients, and 7 of 12 anticardiolipin antibody positive patients, demonstrated reduced A5 binding in this assay, while 10 of 10 healthy blood donor controls did not. Dilution of the aPL sera resulted in progressive increase of A5 binding. Reduction of A5 binding appeared to correlate with reduced A5 anticoagulant activity (r=0.42, n=33, p=.01). This assay shows promise for investigating the effects of aPL antibodies on A5 binding and for the clinical diagnosis of the aPL syndrome. Download : Download high-res image (186KB) Download : Download full-size image Figure Download : Download high-res image (184KB) Download : Download full-size image Figure