Ischemic disorders of the heart can cause an irreversible loss of cardiomyocytes resulting in a substantial decrease of cardiac output. The therapy of choice is heart transplantation, a technique that is hampered by the low number of donor organs. In the present study, we describe the specific labeling, rapid but gentle purification and characterization of cardiomyocytes derived from mouse pluripotent embryonic stem (ES) cells. To isolate the subpopulation of ventricular-like cardiomyocytes, ES cells were stable transfected with the enhanced green fluorescent protein (EGFP) under transcriptional control of the ventricular-specific 2.1 kb myosin light chain-2v (MLC-2v) promoter and the 0.5 kb enhancer element of the cytomegalovirus (CMV(enh).). First fluorescent cells were detected at day 6 + 8 of differentiation within EBs. Four weeks after initiation of differentiation 25% of the cardiomyocyte population displayed fluorescence. Immunohistochemistry revealed the exclusive cardiomyogenic nature of EGFP-positive cells. This was further corroborated by electrophysiological studies where preferentially ventricular phenotypes, but no pacemaker-like cardiomyocytes, were detected among the EGFP-positive population. The enzymatic digestion of EBs, followed by Percoll gradient centrifugation and fluorescence-activated cell sorting, resulted in a 97% pure population of cardiomyocytes. Based on this study, ventricular-like cardiomyocytes can be generated in vitro from EBs and labeled using CMV(enh)./MLC-2v-driven marker genes facilitating an efficient purification. This method may become an important tool for future cell replacement therapy of ischemic cardiomyopathy especially after the proof of somatic differentiation of human ES cells in vitro.

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Key Points• Inhibition of Myb activity by a small molecule blocks proliferation of AML cells and prolongs survival of mice in an in vivo AML model.The transcription factor Myb plays a key role in the hematopoietic system and has been implicated in the development of leukemia and other human cancers. Inhibition of Myb is therefore emerging as a potential therapeutic strategy for these diseases. However, because of a lack of suitable inhibitors, the feasibility of therapeutic approaches based on Myb inhibition has not been explored. We have identified the triterpenoid Celastrol as a potent low-molecular-weight inhibitor of the interaction of Myb with its cooperation partner p300. We demonstrate that Celastrol suppresses the proliferative potential of acute myeloid leukemia (AML) cells while not affecting normal hematopoietic progenitor cells. Furthermore, Celastrol prolongs the survival of mice in a model of an aggressive AML. Overall, our work demonstrates the therapeutic potential of a small molecule inhibitor of the Myb/p300 interaction for the treatment of AML and provides a starting point for the further development of Myb-inhibitory compounds for the treatment of leukemia and, possibly, other tumors driven by deregulated Myb. (Blood. 2016;127(9):1173-1182 Introduction Myb, the protein encoded by the MYB proto-oncogene, is now recognized as an attractive therapeutic target for the treatment of leukemia and potentially for other human tumors.1 Myb was originally discovered as the cellular progenitor of the transforming v-Myb transduced by avian myeloblastosis virus. 2,3 Myb is expressed in the hematopoietic progenitor cells, where it acts as a transcription factor to control genes important for lineage determination, cell proliferation, and differentiation. 4,5 The analysis of Myb null and conditional knockout mice and of mice bearing hypomorphic Myb alleles has demonstrated that Myb is essential for most hematopoietic lineages. [6][7][8][9][10][11] Myb is also expressed in several nonhematopoietic tissues, 12 such as the colonic crypts, where it controls the proliferation and differentiation of the intestinal stem cells. 13 Recent work has shown that deregulated Myb plays critical roles in leukemias and other types of cancer. Recurrent translocations and duplications of the Myb locus occur in acute lymphoblastic leukemia of young children. [14][15][16] In addition, genomic rearrangements of Myb have been reported in acute myelomonocytic and basophilic leukemia. [17][18][19] Although such rearrangements are relatively rare, they indicate that aberrant Myb expression contributes to the development of leukemia. Importantly, it has now been realized that Myb also plays essential roles in leukemias caused by genetic lesions of other genes, such as leukemias driven by human acute myeloid leukemia (AML) oncogenes. [20][21][22][23][24][25][26] High expression of Myb is a common characteristic of these leukemias and is essential for maintenance of the leukemic cells. This was initially observed in studies with Myb ant...

Nowadays patients receiving blood components are exposed to much less transfusion-transmitted infectious diseases than three decades before when among others HIV was identified as causative agent for the acquired immunodeficiency syndrome and the transmission by blood or coagulation factors became evident. Since that time the implementation of measures for risk prevention and safety precaution was socially and politically accepted. Currently emerging pathogens like arboviruses and the well-known bacterial contamination of platelet concentrates still remain major concerns of blood safety with important clinical consequences, but very rarely with fatal outcome for the blood recipient. In contrast to the well-established pathogen inactivation strategies for fresh frozen plasma using the solvent-detergent procedure or methylene blue and visible light, the bench-to-bedside translation of novel pathogen inactivation technologies for cell-containing blood components such as platelets and red blood cells are still underway. This review summarizes the pharmacological/toxicological assessment and the inactivation efficacy against viruses, bacteria, and protozoa of each of the currently available pathogen inactivation technologies and highlights the impact of the results obtained from several randomized clinical trials and hemovigilance data. Until now in some European countries pathogen inactivation technologies are in in routine use for single-donor plasma and platelets. The invention and adaption of pathogen inactivation technologies for red blood cell units and whole blood donations suggest the universal applicability of these technologies and foster a paradigm shift in the manufacturing of safe blood.

IntroductionThe risk of transfusion-transmitted hepatitis B virus (HBV) infection has been reduced by screening all blood donations for HBV surface antigen (HBsAg) since 1970. It was generally accepted that the disappearance of HBsAg indicates the clearance of HBV. Meanwhile, many reports on positive findings for HBV DNA in the liver and blood of HBsAg-negative individuals positive for antibodies against HBV core antigen (anti-HBc) and/or HBsAg (anti-HBs) have been published. 1-8 Blum et al described, in a patient with HBsAg-negative chronic hepatitis who was positive for anti-HBc, anti-HBs, and antibodies against HBe antigen (anti-HBe), a latent HBV infection in hepatocytes with extrachromosomal presence of a full-length viral genome. 9 Michalak et al demonstrated the long-term persistence of HBV DNA in serum and peripheral blood mononuclear cells of patients up to 70 months after complete clinical, biochemical, and serologic recovery from acute viral hepatitis. 10 Rehermann et al showed that traces of HBV were often detectable in the blood for many years after clinical recovery from acute hepatitis, despite the presence of serum antibodies and HBV-specific cytotoxic T lymphocytes (CTLs). 11 These findings suggest that sterilizing immunity to HBV frequently fails to occur and that traces of virus can maintain the CTL response for decades, apparently creating a negative feedback loop that keeps the virus under control, perhaps for life. This was supported by Pasquetto et al, who showed that cytoplasmic HBV nucleocapsids and their cargo of replicative DNA intermediates survive CTL-induced apoptosis of hepatocytes in vitro, 12 and by other groups that demonstrated ongoing viral replication in the liver tissue of patients and healthy individuals after loss of HBsAg. [13][14][15] Furthermore, reactivation of apparently cured HBV infection has been described under chemotherapy or immunomodulating therapy after renal and bone marrow transplantation, and in some of these cases a reverse seroconversion from anti-HBs to HBsAg has been observed. [16][17][18][19] The residual risk of posttransfusion HBV infection has been calculated by several groups in the United States and Germany on the basis of HBV incidence data and the duration of the early window period until HBsAg becomes detectable to be 1:63 000 and less than 1:100 000 blood donations, respectively. 20,21 It has been shown that blood donations of HBsAg-and anti-HBs-negative but anti-HBc-positive HBV carriers can cause posttransfusion hepatitis B. 22;23 Thus, Mosley et al suggested that anti-HBc screening of blood donations might prevent HBV transmission from HBsAgnegative blood donors and that donors positive for anti-HBs as well should be considered noninfectious for HBV. 24 The feasibility of routine polymerase chain reaction (PCR) screening of blood donations in a blood bank setting has been shown by Roth et al. 25 In Germany, the Paul Ehrlich Institute (PEI, Langen, Germany), the institute that defines German Drug Law and is responsible for specific regulati...