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

Alzheimer's disease is a severe irreversible syndrome, characterized by a slow and progressive cognitive decline that interferes with the standard instrumental and essential functions of daily life. Promptly identifying the impairment of particular cognitive functions could be a fundamental condition to limit, through preventive or therapeutic interventions, the functional damages found in this degenerative dementia. This study aims to analyse, through a systematic review of the studies, the sensitivity of four experimental paradigms (Wisconsin Card Sorting Test, Stroop Task, Go/No-Go Task, and Flanker Task) considered as golden standard instruments for executive functions assessment in elderly subjects affected by Alzheimer dementia. This review was carried out according to the PRISMA method. Forty-five studies comparing the executive performance of patients with Alzheimer's dementia (diagnosed according to different classification criteria for dementia) and healthy elderly patients both over the age of sixty, were selected. For the research, PubMed, PsycINFO, PsycArticles databases were used. The study highlighted the importance of using standard protocols to evaluate executive dysfunction in Alzheimer's disease. The Stroop task allows discriminating better between healthy and pathological aging.

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.

Summary:From July 1995 to December 2001, 42 patients with leukemia aged 1-42 years underwent cord blood transplant (CBT) from unrelated, p2 antigen HLA mismatched donors. In all, 26 patients were in p2nd complete remission and 16 in more advanced phase. Conditioning regimens, graft-versus-host disease (GVHD) prophylaxis and supportive policy were uniform for all patients. The cumulative incidence of engraftment was 90% (95% CI: 0.78-0.91). The cumulative incidence of III-IV grade acute-and chronic-GVHD was 9% (95% CI: 0.04-0.24) and 35% (95% CI: 0.21-0.60), respectively. The 4-year cumulative incidence of transplant-related mortality (TRM) and relapse was 28% (95% CI: 0.17-0.47) and 25% (95% CI: 0.14-0.45), respectively. The 4-year overall survival (OS), leukemia-free survival (LFS) and event-free survival (EFS) were 45% (95% CI: 0.27-0.63), 47% (95% CI: 0.30-0.64) and 46% (95% CI: 0.30-0.62), respectively. In multivariate analysis, the most important factor affecting outcomes was the CFU-GM dose, associated with CMV serology (P ¼ 0.003 and 0.04, respectively) in influencing OS and with patient sex (P ¼ 0.008 and 0.03, respectively) in influencing LFS. Finally, CFU-GM dose was the only factor that affected EFS significantly (P ¼ 0.02). In conclusion, the infused cell dose expressed as in vitro progenitor cell growth is highly predictive of outcomes after an unrelated CBT and should be considered the main parameter in selecting cord blood units for transplant.