Key Points• Haploidentical, unmanipulated, G-CSF-primed bone marrow transplantation.• Haploidentical hematopoietic stem cell transplantation for hematologic malignancies.Eighty patients with high-risk hematologic malignancies underwent unmanipulated, G-CSF-primed BM transplantation from an haploidentical family donor. Patients were transplanted in first or second complete remission (CR, standard-risk: n ‫؍‬ 45) or in > second CR or active disease (high-risk: n ‫؍‬ 35). The same regimen for GVHD prophylaxis was used in all cases. The cumulative incidence (CI) of neutrophil engraftment was 93% ؎ 0.1%. The 100-day CIs for II-IV and III-IV grade of acute GVHD were 24% ؎ 0.2% and 5% ؎ 0.6%, respectively. The 2-year CI of extensive chronic GVHD was 6% ؎ 0.1%. The 1-year CI of treatment-related mortality was 36% ؎ 0.3%. After a median follow-up of 18 months, 36 of 80 (45%) patients are alive in CR. The 3-year probability of overall and disease-free survival for standard-risk and high-risk patients was 54% ؎ 8% and 33% ؎ 9% and 44% ؎ 8% and 30% ؎ 9%, respectively. In multivariate analysis, disease-free survival was significantly better for patients who had standard-risk disease and received transplantations after 2007. We conclude that unmanipulated, G-CSF-primed BM transplantation from haploidentical family donor provides very encouraging results in terms of engraftment rate, incidence of GVHD and survival and represents a feasible, valid alternative for patients with high-risk malignant hematologic diseases, lacking an HLA identical sibling and in need to be urgently transplanted. (Blood. 2013;121(5): 849-857)

Ex vivo amplification of human hematopoietic stem cells (HSC) without loss of their self-renewing potential represents an important target for transplantation, gene and cellular therapies. Valproic acid is a safe and widely used neurologic agent that acts as a potent inhibitor of histone deacetylase activities. Here, we show that valproic acid addition to liquid cultures of human CD34+ + cells isolated from cord blood, mobilized peripheral blood, and bone marrow strongly enhances the ex vivo expansion potential of different cytokine cocktails as shown by morphologic, cytochemical, immunophenotypical, clonogenic, and gene expression analyses. Notably, valproic acid highly preserves the CD34 positivity after 1 week (range, 40-89%) or 3 weeks (range, 21-52%) amplification cultures with two (Flt3L + + thrombopoietin) or four cytokines (Flt3L + + thrombopoietin + + stem cell factor + + interleukin 3). Moreover, valproic acid treatment increases histone H4 acetylation levels at specific regulatory sites on HOXB4, a transcription factor gene with a key role in the regulation of HSC self-renewal and AC133, a recognized marker gene for stem cell populations. Overall, our results relate the changes induced by valproic acid on chromatin accessibility with the enhancement of the cytokine effect on the maintenance and expansion of a primitive hematopoietic stem cell population. These findings underscore the potentiality of novel epigenetic approaches to modify HSC fate in vitro. (Cancer Res 2005; 65(4): 1505-13)

BackgroundPersistent mixed chimerism represents a state in which recipient and donor cells stably co-exist after hematopoietic stem cell transplantation. However, since in most of the studies reported in literature the engraftment state was observed in the nucleated cells, in this study we determined the donor origin of the mature erythrocytes of patients with persistent mixed chimerism after transplantation for hemoglobinopathies. Results were compared with the engraftment state observed in singly picked out burst-forming unit -erythroid colonies and in the nucleated cells collected from the peripheral blood and from the bone marrow. Design and MethodsThe donor origin of the erythrocytes was determined analyzing differences on the surface antigens of the erythrocyte suspension after incubation with anti-ABO and/or anti-C, -c, -D, -E and -e monoclonal antibodies by a flow cytometer. Analysis of short tandem repeats was used to determine the donor origin of nucleated cells and burst-forming unit -erythroid colonies singly picked out after 14 days of incubation. ResultsThe proportions of donor-derived nucleated cells in four transplanted patients affected by hemoglobinopathies were 71%, 46%, 15% and 25% at day 1364, 1385, 1314 and 932, respectively. Similar results were obtained for the erythroid precursors, analyzing the donor/recipient origin of the burst-forming unit -erythroid colonies. In contrast, on the same days of observation, the proportions of donor-derived erythrocytes in the four patients with persistent mixed chimerism were 100%, 100%, 73% and 90%. ConclusionsOur results showed that most of the erythrocytes present in four long-term transplanted patients affected by hemoglobinopathies and characterized by the presence of few donor engrafted nucleated cells were of donor origin. The indication that small proportions of donor engrafted cells might be sufficient for clinical control of the disease in patients affected by hemoglobinopathies is relevant, although the biological mechanisms underlying these observations need further investigation.Key words: hemoglobinopathies, persistent mixed chimerism, hematopoietic stem cell transplantation.Citation: Andreani M, Testi M, Gaziev J, Condello R, Bontadini A, Tazzari PL, Ricci F, De Felice L, Agostini F, Fraboni D, Ferrari G, Battarra M, Troiano M, Sodani P and Lucarelli G. Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease. Haematologica 2011;96(1):128-133. doi:10.3324/haematol.2010 This is an open-access paper.Quantitatively different red cell/nucleated cell chimerism in patients with long-term, persistent hematopoietic mixed chimerism after bone marrow transplantation for thalassemia major or sickle cell disease

Osteoclasts from a patient affected by osteopetrosis were examined in vivo and in vitro. Iliac crest biopsy revealed an osteosclerotic pattern, with prominent numbers of osteoclasts noted for hypernuclearity and incomplete adherence to the bone surface. A population comprising tartrate-resistant acid phosphatase (TRAP)-positive, multinucleated and mononuclear cells, and alkaline phosphatase-positive stromal fibroblasts was obtained in vitro from bone marrow. Mononuclear TRAP-positive precursors spontaneously fused in culture to form giant osteoclast-like cells. These cells expressed the osteoclast marker MMP-9 and calcitonin receptor, and lacked the macrophage marker, Fc receptor. Expression and distribution of c-src, c-fms, and CD68, and response to steroid hormones relevant to osteoclast differentiation and function were apparently normal, whereas cell retraction in response to calcitonin was impaired. TRAP-positive multinucleated cells did not form osteoclast-specific adhesion structures (clear zone, podosomes, or actin rings). Bone resorption rate was severely reduced in vitro. Focal adhesions and stress fibers were observed en lieu of podosomes and actin rings. Adhesion structures contained low levels of immunoreactive vitronectin receptor, most of this integrin being retained in cytoplasmic vesicles. These data provide the first characterization of abnormal differentiation and function of human osteopetrotic osteoclastlike

Summary. Umbilical cord blood (UCB) has been successfully used for haemopoietic stem cell transplantation, although its use has been cautiously limited to paediatric patients because of the reduced volume produced. The clinical results have con®rmed that either engraftment or survival signi®cantly correlate with cell dose infused. We have standardized a culture method providing in a short time a signi®cant ampli®cation of both committed progenitors and primitive stem cells for clinical use.Eight-day culture of UCB cells with¯t3L/SCF/PIXY 321 induced a 10-fold ampli®cation of CD34 cells and the expansion of multipotent (CFU-GEMM) and committed (CFU-GM, BFU-E) progenitors respectively of 5-, 7-and 9-fold over input cells. As to the early stem cell pool, the primitive CD34 Thy-1 cell fraction increased 6-fold and the LTC-IC were ampli®ed 17-fold. Furthermore, the in vitro proliferation was detected by the gradual loss of¯uorescence of the CD34 cells tracked at day 0 with the dye PKH26. After 8 d of ampli®cation >6% of the CD34 cells remained intensely¯uorescent. This subpopulation represents a deeply quiescent cell fraction unresponsive to cytokines and very enriched of primitive stem cells. These cells are most likely to be responsible for long-term reconstitution after transplant.