Identification of the niche and phenotype of the first human hematopoietic stem cells

SummaryIn various vertebrate species, the dorsal aorta (Ao) is the site of specification of adult hematopoietic stem cells (HSCs). It has been observed that the upregulation of essential hematopoietic transcription factors and the formation of specific intra-aortic hematopoietic cell clusters occur predominantly in the ventral domain of the Ao (AoV). In the mouse, the first HSCs emerge in the AoV. Here, we demonstrate that in the human embryo the first definitive HSCs also emerge asymmetrically and are localized to the AoV, which thus identifies a functional niche for developing human HSCs. Using magnetic cell separation and xenotransplantations, we show that the first human HSCs are CD34+VE-cadherin+CD45+C-KIT+THY-1+Endoglin+RUNX1+CD38−/loCD45RA−. This population harbors practically all committed hematopoietic progenitors and is underrepresented in the dorsal domain of the Ao (AoD) and urogenital ridges (UGRs). The present study provides a foundation for analysis of molecular mechanisms underpinning embryonic specification of human HSCs

Hemogenic Endothelial Cells Can Transition to Hematopoietic Stem Cells through a B-1 Lymphocyte-Biased State during Maturation in the Mouse Embryo

Precursors of hematopoietic stem cells (pre-HSCs) have been identified as intermediate precursors during the maturation process from hemogenic endothelial cells to HSCs in the aorta-gonad-mesonephros (AGM) region of the mouse embryo at embryonic day 10.5. Although pre-HSCs acquire an efficient adult-repopulating ability after ex vivo co-culture, their native hematopoietic capacity remains unknown. Here, we employed direct transplantation assays of CD45-VE-cadherin(VC)+KIT+(V+K+) cells (containing pre-HSCs) into immunodeficient neonatal mice that permit engraftment of embryonic hematopoietic precursors. We found that freshly isolated V+K+ cells exhibited significantly greater B-1 lymphocyte-biased repopulating capacity than multilineage repopulating capacity. Additionally, B cell colony-forming assays demonstrated the predominant B-1 progenitor colony-forming ability of these cells; however, increased B-2 progenitor colony-forming ability emerged after co-culture with Akt-expressing AGM endothelial cells, conditions that support pre-HSC maturation into HSCs. Our studies revealed an unexpected B-1 lymphocyte bias of the V+K+ population and acquisition of B-2 potential during commitment to the HSC fate

Neuroprotection in a Novel Mouse Model of Multiple Sclerosis

The authors acknowledge the support of the Barts and the London Charity, the Multiple Sclerosis Society of Great Britain and Northern Ireland, the National Multiple Sclerosis Society, USA, notably the National Centre for the Replacement, Refinement & Reduction of Animals in Research, and the Wellcome Trust (grant no. 092539 to ZA). The siRNA was provided by Quark Pharmaceuticals. The funders and Quark Pharmaceuticals had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Dielectric Spectroscopy of Biological Samples

For the first time, the impedance spectrum of live Jurkat T-lymphocytes human cells was characterized in a single sweep spanning six decades of frequency from 9 kHz to 9 GHz. The ultrawide bandwidth bridged the traditional impedance spectroscopy at kilohertz to megahertz frequencies with the recently developed microwave dielectric spectroscopy, which can probe the cell interior without being hindered by the cell membrane. Based on the measured scattering parameters and a simple cell model, an equivalent circuit of four nondispersive elements, including membrane resistance, membrane capacitance, cytoplasm resistance, and cytoplasm capacitance, was extracted and found sufficient to explain the so-called β relaxation over the frequencies measured. The extracted cell parameters were in general agreement with the literature. However, the presently extracted membrane capacitance of 0.4 pF and cytoplasm resistance of 0.75 MΩ are on the low and high end of the literature, respectively. This could be explained by having separated out the shunt effects of the membrane resistance and cytoplasm capacitance, respectively. In fact, the present membrane resistance and cytoplasm capacitance, at 2.8 MΩ and 10 fF, respectively, are believed to be more reliable due to the low-conductivity solution and the microwave frequency used. Meanwhile, sensitivity analysis was carried out for extracting lumped cell characteristics such as membrane resistance and cytoplasm capacitance from the scattering parameters. The scattering parameters were measured on a coplanar waveguide with a Jurkat cell trapped by dielectrophoresis either in a series or shunt configuration. The sensitivity analysis validated our previous empirical observation that the insertion loss of a series-trapped cell and the return loss of a shunt-trapped cell were most sensitive to the cell impedance. Additionally, the membrane resistance and cytoplasm capacitance were most sensitive to low- and high-frequency scattering parameters, respectively. Furthermore, the dissertation presents a novel in situ single-connection calibration using biocompatible solutions, which is demonstrated in single-cell characterization from 0.5 GHz to 9 GHz for the first time as well. The characterization is based on quickly trapping and detrapping the cell by dielectrophoresis on a coplanar waveguide (CPW) with a small protrusion in one of its ground electrodes, which doubles as the calibration standard when covered by different liquids. Consistent with theoretical analysis, the difference in the transmission coefficient increases with increasing frequency and is generally smaller than the difference in the reflection coefficient. With improved accuracy and throughput, the calibration technique may enable broadband electrical characterization of single cells in a high-speed cytometer

Rust resistance in faba bean (Vicia faba L.) : status and strategies for improvement

Faba bean (Vicia faba L.) is an important grain legume used as food and feed. Its production is threatened by abiotic stresses and diseases, of which rust (Uromyces viciae-fabae) is one of the major diseases in East and North Africa, China and the northern grain growing region of Australia. Understanding the genetic and physiological mechanisms of rust resistance in faba bean is in an early phase. The presence of seedling and adult plant resistance genes has been observed. The resistance most frequently utilised in applied plant breeding is race-specific, where the interaction between resistance genes in the host and avirulence genes in the pathogen confers resistance. The main drawback of using race-specific resistance is lack of durability, when deployed singly. Slow rusting or partial resistance, controlled by multiple genes of small effect, is generally non-race specific, so it can be more durable. We present the current knowledge of host resistance and pathogen diversity and propose rational breeding approaches aided with molecular markers to breed durable rust resistance in faba bean.Peer reviewe

Structured near-infrared Magnetic Circular Dichroism spectra of the Mn₄CaO₅ cluster of PSII in T. vulcanus are dominated by Mn(IV) d-d 'spin-flip' transitions

Photosystem II passes through four metastable S-states in catalysing light-driven water oxidation. Variable temperature variable field (VTVH) Magnetic Circular Dichroism (MCD) spectra in PSII of Thermosynochococcus (T.) vulcanus for each S-state are reported. These spectra, along with assignments, provide a new window into the electronic and magnetic structure of Mn₄CaO₅. VTVH MCD spectra taken in the S₂state provide a clear g=2, S=1/2 paramagnetic characteristic, which is entirely consistent with that known by EPR. The three features, seen as positive (+) at 749nm, negative (-) at 773nm and (+) at 808nm are assigned as ⁴A→²E spin-flips within the d³ configuration of the Mn(IV) centres present. This assignment is supported by comparison(s) to spin-flips seen in a range of Mn(IV) materials. S₃ exhibits a more intense (-) MCD peak at 764nm and has a stronger MCD saturation characteristic. This S₃ MCD saturation behaviour can be accurately modelled using parameters taken directly from analyses of EPR spectra. We see no evidence for Mn(III) d-d absorption in the near-IR of any S-state. We suggest that Mn(IV)-based absorption may be responsible for the well-known near-IR induced changes induced in S₂ EPR spectra of T. vulcanus and not Mn(III)-based, as has been commonly assumed. Through an analysis of the nephelauxetic effect, the excitation energy of S-state dependent spin-flips seen may help identify coordination characteristics and changes at each Mn(IV). A prospectus as to what more detailed S-state dependent MCD studies promise to achieve is outlined.We recognise the support of the Australian Research Council through grants DP110104565 and DP150103137 (E.K.), FT140100834 (N.C) and MEXT/JSPS of Japan through a Grant-in-Aid for Specially Promoted Research No. 24000018 (J.R.S.)

EPR spectroscopy of iron- and nickel-doped [ZnAl]-layered double hydroxides: modeling active sites in heterogeneous water oxidation catalysts

Iron-doped nickel layered double hydroxides (LDHs) are among the most active heterogeneous water oxidation catalysts. Due to inter-spin interactions, however, the high density of magnetic centers results in line-broadening in magnetic resonance spectra. As a result, gaining atomic-level insight into the catalytic mechanism via electron paramagnetic resonance (EPR) is not generally possible. To circumvent spin-spin broadening, iron and nickel atoms were doped into non-magnetic [ZnAl]-LDH materials and the coordination environments of the isolated Fe(III) and Ni(II) sites were characterized. Multifrequency EPR spectroscopy identified two distinct Fe(III) sites (S = 5/2) in [Fe:ZnAl]-LDH. Changes in zero field splitting (ZFS) were induced by dehydration of the material, revealing that one of the Fe(III) sites is solvent-exposed (i.e. at an edge, corner, or defect site). These solvent-exposed sites feature an axial ZFS of 0.21 cm⁻¹ when hydrated. The ZFS increases dramatically upon dehydration (to -1.5 cm⁻¹), owing to lower symmetry and a decrease in the coordination number of iron. The ZFS of the other (“inert”) Fe(III) site maintains an axial ZFS of 0.19-0.20 cm⁻¹ under both hydrated and dehydrated conditions. We observed a similar effect in [Ni:ZnAl]-LDH materials; notably, Ni(II) (S = 1) atoms displayed a single, small ZFS (±0.30 cm⁻¹) in hydrated material, whereas two distinct Ni(II) ZFS values (±0.30 and ±1.1 cm⁻¹) were observed in the dehydrated samples. Although the magnetically-dilute materials were not active catalysts, the identification of model sites in which the coordination environments of iron and nickel were particularly labile (e.g. by simple vacuum drying) is an important step towards identifying sites in which the coordination number may drop spontaneously in water, a probable mechanism of water oxidation in functional materials