Systematic investigation of oxygen and growth factors in clinically valid ex vivo expansion of cord blood CD34+ hematopoietic progenitor cells

Background aims. Cord blood is considered to be a superior source of hematopoietic stem and progenitor cells for transplantation, but clinical use is limited primarily because of the low numbers of cells harvested. Ex vivo expansion has the potential to provide a safe, effective means of increasing cell numbers. However, an absence of consensus regarding optimum expansion conditions prevents standard implementation. Many studies lack clinical applicability, or have failed to investigate the combinational effects of different parameters. Methods. This is the first study to characterize systematically the effect of growth factor combinations across multiple oxygen levels on the ex vivo expansion of cord blood CD34 hematopoietic cells utilizing clinically approvable reagents and methodologies throughout. Results. Optimal fold expansion, as assessed both phenotypically and functionally, was greatest with thrombopoietin, stem cell factor, Flt-3 ligand and interleukin-6 at an oxygen level of 10%. With these conditions, serial expansion showed continual target population expansion and consistently higher expression levels of self-renewal associated genes. Conclusions. This study has identified optimized fold expansion conditions, with the potential for direct clinical translation to increase transplantable cell dose and as a baseline methodology against which future factors can be tested.<br /

Direct comparison of four hematopoietic differentiation methods from human induced pluripotent stem cells

Tursky and colleagues addressed a vital, unmet need to aid method selection for iPSC hematopoietic differentiation for research and potentially clinical use, comparing four representative serum- and feeder-layer-free methods. Assessments include production efficiency of CD34 and functional hematopoietic progenitor cells, cost-benefit analysis, and ability to recapitulate aberrant hematopoiesis. An optimized method proved the most efficient, cost-effective, and sensitive.Induced pluripotent stem cells (iPSCs) are an invaluable resource for the study of human disease. However, there are no standardized methods for differentiation into hematopoietic cells, and there is a lack of robust, direct comparisons of different methodologies. In the current study we improved a feeder-free, serum-free method for generation of hematopoietic cells from iPSCs, and directly compared this with three other commonly used strategies with respect to efficiency, repeatability, hands-on time, and cost. We also investigated their capability and sensitivity to model genetic hematopoietic disorders in cells derived from Down syndrome and β-thalassemia patients. Of these methods, a multistep monolayer-based method incorporating aryl hydrocarbon receptor hyperactivation (“2D-multistep”) was the most efficient, generating significantly higher numbers of CD34 progenitor cells and functional hematopoietic progenitors, while being the most time- and cost-effective and most accurately recapitulating phenotypes of Down syndrome and β-thalassemia

Do phytogeographic patterns reveal biomes or biotic regions?

We present the largest comparative biogeographical analysis that has complete coverage of Australia's geography (20 phytogeographical subregions), using the most complete published molecular phylogenies to date of large Australian plant clades (Acacia, Banksia and the eucalypts). Two distinct sets of areas within the Australian flora were recovered, using distributional data from the Australasian Virtual Herbarium (AVH) and the Atlas of Living Australia (ALA): younger Temperate, Eremaean and Monsoonal biomes, and older southwest + west, southeast and northern historical biogeographical regions. The analyses showed that by partitioning the data into two sets, using either a Majority or a Frequency method to select taxon distributions, two equally valid results were found. The dataset that used a Frequency method discovered general area cladograms that resolved patterns of the Australian biomes, whereas if widespread taxa (Majority method, with &gt;50% of occurrences outside a single subregion) were removed the analysis then recovered historical biogeographical regions. The study highlights the need for caution when processing taxon distributions prior to analysis as, in the case of the history of Australian phytogeography, the validity of both biomes and historical areas have been called into question.50% of occurrences outside a single subregion) were removed the analysis then recovered historical biogeographical regions. The study highlights the need for caution when processing taxon distributions prior to analysis as, in the case of the history of Australian phytogeography, the validity of both biomes and historical areas have been called into question

Heart and Liver Defects and Reduced Transforming Growth Factor β2 Sensitivity in Transforming Growth Factor β Type III Receptor-Deficient Embryos

The type III transforming growth factor β (TGFβ) receptor (TβRIII) binds both TGFβ and inhibin with high affinity and modulates the association of these ligands with their signaling receptors. However, the significance of TβRIII signaling in vivo is not known. In this study, we have sought to determine the role of TβRIII during development. We identified the predominant expression sites of ΤβRIII mRNA as liver and heart during midgestation and have disrupted the murine TβRIII gene by homologous recombination. Beginning at embryonic day 13.5, mice with mutations in ΤβRIII developed lethal proliferative defects in heart and apoptosis in liver, indicating that TβRIII is required during murine somatic development. To assess the effects of the absence of TβRIII on the function of its ligands, primary fibroblasts were generated from TβRIII-null and wild-type embryos. Our results indicate that TβRIII deficiency differentially affects the activities of TGFβ ligands. Notably, TβRIII-null cells exhibited significantly reduced sensitivity to TGFβ2 in terms of growth inhibition, reporter gene activation, and Smad2 nuclear localization, effects not observed with other ligands. These data indicate that TβRIII is an important modulator of TGFβ2 function in embryonic fibroblasts and that reduced sensitivity to TGFβ2 may underlie aspects of the TβRIII mutant phenotype

Integrative Genomics Identifies the Molecular Basis of Resistance to Azacitidine Therapy in Myelodysplastic Syndromes

Myelodysplastic syndromes and chronic myelomonocytic leukemia are blood disorders characterized by ineffective hematopoiesis and progressive marrow failure that can transform into acute leukemia. The DNA methyltransferase inhibitor 5-azacytidine (AZA) is the most effective pharmacological option, but only ∼50% of patients respond. A response only manifests after many months of treatment and is transient. The reasons underlying AZA resistance are unknown, and few alternatives exist for non-responders. Here, we show that AZA responders have more hematopoietic progenitor cells (HPCs) in the cell cycle. Non-responder HPC quiescence is mediated by integrin α5 (ITGA5) signaling and their hematopoietic potential improved by combining AZA with an ITGA5 inhibitor. AZA response is associated with the induction of an inflammatory response in HPCs in vivo. By molecular bar coding and tracking individual clones, we found that, although AZA alters the sub-clonal contribution to different lineages, founder clones are not eliminated and continue to drive hematopoiesis even in complete responders