Gravitational field-flow fractionation of human hemopoietic stem cells

New cell sorting methodologies, which are simple, fast, non-invasive, and able to isolate homogeneous cell populations, are needed for applications ranging from gene expression analysis to cell-based therapy. In particular, in the forefront of stem cell isolation, progenitor cells have to be separated under mild experimental conditions from complex heterogeneous mixtures prepared from human tissues. Most of the methodologies now employed make use of immunological markers. However, it is widely acknowledged that specific markers for pluripotent stem cells are not as yet available, and cell labelling may interfere with the differentiation process. This work presents for the first time gravitational field-flow fractionation (GrFFF), as a tool for tag-less, direct selection of human hematopoietic stem and progenitor cells from cell samples obtained by peripheral blood aphaeresis. These cells are responsible to repopulate the hemopoietic system and they are used in transplantation therapies. Blood aphaeresis sample were injected into a GrFFF system and collected fractions were characterized by flow cytometry for CD34 and CD45 expression, and then tested for viability and multi-differentiation potential. The developed GrFFF method allowed obtaining high enrichment levels of viable, multi-potent hematopoietic stem cells in specific fraction and it showed to fulfil major requirements of analytical performance, such as selectivity and reproducibility of the fractionation process and high sample recovery.     

Osteosarcoma Stem Cell Potent Gallium(III)-Polypyridyl Complexes Bearing Diflunisal

We report the anti-osteosarcoma stem cell (OSC) properties of a series of gallium(III)-polypyridyl complexes ( 5 - 7 ) containing diflunisal, a non-steroidal anti-inflammatory drug. The most effective complex within the series, 6 (containing 3,4,7,8-tetramethyl-1,10-phenanthroline), displayed similar potency towards bulk osteosarcoma cells and OSCs, in the nanomolar range. Remarkably, 6 exhibited significantly higher monolayer and sarcosphere OSC potency (up to three orders of magnitude) than clinically approved drugs used in frontline (cisplatin and doxorubicin) and secondary (etoposide, ifosfamide, and carboplatin) osteosarcoma treatments. Mechanistic studies show that 6 downregulates cyclooxygenase-2 (COX-2) and kills osteosarcoma cells in a COX-2 dependent manner. Furthermore, 6 induces genomic DNA damage and caspase-dependent apoptosis. To the best of our knowledge, 6 is the first metal complex to kill osteosarcoma cells by simultaneously inhibiting COX-2 and damaging nuclear DNA.     

Preparation of FeS/iron oxide composite and flax stem‐based porous carbon and its Cr(VI) removal ability

Using flax stem and ferrous sulfate, a composite porous carbon material was prepared by means of high‐temperature roasting and a one‐step process in a muffle furnace. The samples were characterized using X‐Ray diffraction (XRD) and Scanning electron microscopy (SEM), and the effects of ferrous sulfate concentration, carbonization temperature, and pH values of Cr(VI) aqueous solution on the removal performance of Cr(VI) were studied. XRD and SEM analysis showed that the prepared samples were amorphous porous carbon loaded with FeS/Fe₂O₃/Fe₃O₄. High FeSO₄ impregnation concentration, high carbonization temperature, and a low pH value of Cr(VI) aqueous solution were beneficial for Cr(VI) removal. When pH = 2, the amount of Cr(VI) removal was 99.93 mg/g by the sample obtained from 1 g flax powder impregnated in 4.5 mmol FeSO₄/40 mL H₂O solution and calcined for 2 hr at 800°C.     

Conductive Biomaterials as Substrates for Neural Stem Cells Differentiation towards Neuronal Lineage Cells

The injuries and defects in the central nervous system are the causes of disability and death of an affected person. As of now, there are no clinically available methods to enhance neural structural regeneration and functional recovery of nerve injuries. Recently, some experimental studies claimed that the injuries in brain can be repaired by progenitor or neural stem cells located in the neurogenic sites of adult mammalian brain. Various attempts have been made to construct biomimetic physiological microenvironment for neural stem cells to control their ultimate fate. Conductive materials have been considered as one the best choices for nerve regeneration due to the capacity to mimic the microenvironment of stem cells and regulate the alignment, growth, and differentiation of neural stem cells. The review highlights the use of conductive biomaterials, e.g., polypyrrole, polyaniline, poly(3,4‐ethylenedioxythiophene), multi‐walled carbon nanotubes, single‐wall carbon nanotubes, graphene, and graphite oxide, for controlling the neural stem cells activities in terms of proliferation and neuronal differentiation. The effects of conductive biomaterials in axon elongation and synapse formation for optimal repair of central nervous system injuries are also discussed.     

Quantifiable correlation of ToF-SIMS and XPS data from polymer surfaces with controlled amino acid and peptide content

Peptide-coated surfaces are widely employed in biomaterial design, but quantifiable correlation between surface composition and biological response is challenging due to, for example, instrumental limitations, a lack of suitable model surfaces or limitations in quantitatively correlating data from different surface analytical techniques. Here, we first establish a reference material that allows control over amino acid content. Reversible addition-fragmentation chain-transfer (RAFT) polymerisation is used to prepare a copolymer containing alkyne and furan units with well-defined chain length and composition. Huisgen Cu(I)-catalysed azide-alkyne cycloaddition reaction is used to attach the model azido-polyethyleneglycol-amide-modified pentafluoro-l -phenylalanine to the polymer. Different compositional ratios of the polymer provide a surface with varying amino acid content that is analysed by X-ray photoelectron spectroscopy (XPS) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). Nitrogen-related signals are compared with fluorine signals from both techniques. Fluorine and nitrogen signals from both techniques are found to be related to the copolymer compositions, but the homopolymer data deviate from this trend. The approach is then translated to a heparin-binding peptide that supports cell adhesion. Human embryonic stem cells cultured on copolymer surfaces presenting different amounts of heparin-binding peptide show strong cell growth while maintaining pluripotency after 72 h of culture. The early cell adhesion at 24 h can be correlated to the logarithm of the normalised CH₄N⁺ ion intensity from ToF-SIMS data, which is established as a suitable and generalisable marker ion for amino acids and peptides. This work contributes to the ability to use ToF-SIMS in a more quantitative manner for the analysis of amino acid and peptide surfaces.     

Compound A Increases Cell Infiltration in Target Organs of Acute Graft-versus-Host Disease (aGVHD) in a Mouse Model

Systemic steroids are used to treat acute graft-versus-host disease (aGVHD) caused by allogenic bone marrow transplantation (allo-BMT); however, their prolonged use results in complications. Hence, new agents for treating aGVHD are required. Recently, a new compound A (CpdA), with anti-inflammatory activity and reduced side effects compared to steroids, has been identified. Here, we aimed to determine whether CpdA can improve the outcome of aGVHD when administered after transplantation in a mouse model (C57BL/6 in B6D2F1). After conditioning with 9Gy total body irradiation, mice were infused with bone marrow (BM) cells and splenocytes from either syngeneic (B6D2F1) or allogeneic (C57BL/6) donors. The animals were subsequently treated (3 days/week) with 7.5 mg/kg CpdA from day +15 to day +28; the controls received 0.9% NaCl. Thereafter, the incidence and severity of aGVHD in aGVHD target organs were analyzed. Survival and clinical scores did not differ significantly; however, CpdA-treated animals showed high cell infiltration in the target organs. In bulk mixed lymphocyte reactions, CpdA treatment reduced the cell proliferation and expression of inflammatory cytokines and chemokines compared to controls, whereas levels of TNF, IL-23, chemokines, and chemokine receptors increased. CpdA significantly reduced proliferation in vitro but increased T cell infiltration in target organs.