Multiple waves of T lymphopoiesis and characterization of hemogenic endothelium in zebrafish

Abstract Abstract 2352 Blood is one of the most highly regenerative tissues and is generated by a rare population of hematopoietic stem cells (HSCs). Generation of HSCs seems to be restricted during early embryonic development to particular fetal tissues, such as yolk sac, aorta gonad mesonephros (AGM) region and placenta. The competence of HSCs to self-renew and differentiate towards multiple lineages sustains the generation of blood cells for an entire lifespan. Human HSCs are defined by their self-renewal and multilineage differentiation in functional xenotransplantation assays. Differentiation of human embryonic stem cells (hESCs) into transplantable HSCs continues to be a major challenge in the field. We believe that generation of functional multipotent HSCs from hESC requires an approach that recapitulates early human development both temporally and spatially. Thus, we have developed an innovative in vitro system that mimics the early events of human embryonic development prior to hematopoietic generation; where 1) induction of a single ESC monolayers to mesodermal fate; 2) single cell mesoderm progenitors are placed into a fetal AGM stromal environment under conditions that favor endothelial differentiation; and 3) hematopoietic inductors are added generating single free-floating hematopoietic progenitors in a temporal fashion. We have identified both hemogenic endothelium (adherent) and hematopoietic (non-adherent) cells in the AGM/mesoderm coculture. Hemogenic endothelium defined by expression of VE-Cadherin cocultured on AFT024 fetal liver stromal cells is able to generate hematopoietic precursors. Non-adherent cells start budding up by day 18 in the AGM/mesoderm coculture. Flow cytometry analysis shows that CD31 is the prevalent marker in this floating population. Non-adherent VE-Caherin-CD31+CD34+ cells arising between day 24 till 33 have shown the highest hematopoietic activity as measure by colony forming units (CFU) cells, cobblestone area-forming cell assays (week 4–5 CAFC) and their potential to generate both B and T-cells. Molecular characterization of hemogenic endothelium the non-adherent VE-Caherin-CD31+CD34+ cells by qRT-PCR confirm that also a temporal gene expression is taken place as cells transit from endothelium to hematopoietic cells. VE-Caherin-CD31+CD34+ cells also express CRCX4, suggesting their potential for engrafting in mice models. This novel approach provides a unique opportunity to study the early events and to identify molecular targets in the human hematopoietic commitment, lineage specification, and maturation; these hematopoietic cells may represent a promise for clinical therapies in a diverse range of blood diseases. Disclosures: No relevant conflicts of interest to declare.

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Morphological Characterization of Mycobacteriophage R1

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100051281 ◽

1974 ◽

Vol 32 ◽

pp. 254-255

Author(s):

B. L. Soloff ◽

T. A. Rado

Keyword(s):

Carbon Source ◽

Stationary Phase ◽

Growth Phase ◽

Minimal Medium ◽

Morphological Characterization ◽

Logarithmic Growth Phase ◽

Complete Lysis ◽

Lysogenic Culture ◽

Logarithmic Growth

Mycobacteriophage R1 was originally isolated from a lysogenic culture of M. butyricum. The virus was propagated on a leucine-requiring derivative of M. smegmatis, 607 leu−, isolated by nitrosoguanidine mutagenesis of typestrain ATCC 607. Growth was accomplished in a minimal medium containing glycerol and glucose as carbon source and enriched by the addition of 80 μg/ ml L-leucine. Bacteria in early logarithmic growth phase were infected with virus at a multiplicity of 5, and incubated with aeration for 8 hours. The partially lysed suspension was diluted 1:10 in growth medium and incubated for a further 8 hours. This permitted stationary phase cells to re-enter logarithmic growth and resulted in complete lysis of the culture.

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AEM analysis of crystallization in Ti-based amorphous alloys

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100075142 ◽

1983 ◽

Vol 41 ◽

pp. 270-271

Author(s):

A.R. Pelton ◽

A.F. Marshall ◽

Y.S. Lee

Keyword(s):

Magnetic Properties ◽

Amorphous Alloys ◽

Amorphous Materials ◽

Isothermal Annealing ◽

Amorphous Matrix ◽

Nucleation And Growth ◽

Current Interest ◽

Amorphous Films ◽

Electrical And Magnetic Properties

Amorphous materials are of current interest due to their desirable mechanical, electrical and magnetic properties. Furthermore, crystallizing amorphous alloys provides an avenue for discerning sequential and competitive phases thus allowing access to otherwise inaccessible crystalline structures. Previous studies have shown the benefits of using AEM to determine crystal structures and compositions of partially crystallized alloys. The present paper will discuss the AEM characterization of crystallized Cu-Ti and Ni-Ti amorphous films.Cu60Ti40: The amorphous alloy Cu60Ti40, when continuously heated, forms a simple intermediate, macrocrystalline phase which then transforms to the ordered, equilibrium Cu3Ti2 phase. However, contrary to what one would expect from kinetic considerations, isothermal annealing below the isochronal crystallization temperature results in direct nucleation and growth of Cu3Ti2 from the amorphous matrix.

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Characterization of Coherent, Ordered Precipitates in Nickel-Base Alloys

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100071168 ◽

1973 ◽

Vol 31 ◽

pp. 144-145

Author(s):

B. H. Kear ◽

J. M. Oblak

Keyword(s):

Stable Phase ◽

Nickel Base Superalloy ◽

Alloy 718 ◽

Commercial Alloy ◽

Precipitate Morphology ◽

Continuous Precipitation ◽

Nickel Base ◽

Base Superalloy ◽

Strengthening Precipitate

A nickel-base superalloy is essentially a Ni/Cr solid solution hardened by additions of Al (Ti, Nb, etc.) to precipitate a coherent, ordered phase. In most commercial alloy systems, e.g. B-1900, IN-100 and Mar-M200, the stable precipitate is Ni3 (Al,Ti) γ′, with an LI2structure. In A lloy 901 the normal precipitate is metastable Nis Ti3 γ′ ; the stable phase is a hexagonal Do2 4 structure. In Alloy 718 the strengthening precipitate is metastable γ″, which has a body-centered tetragonal D022 structure.Precipitate MorphologyIn most systems the ordered γ′ phase forms by a continuous precipitation re-action, which gives rise to a uniform intragranular dispersion of precipitate particles. For zero γ/γ′ misfit, the γ′ precipitates assume a spheroidal.

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The Use of Advanced Analytical Techniques for Characterization of the Chemical, Physical, and Crystallographic Nature of a Surface

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100071107 ◽

1973 ◽

Vol 31 ◽

pp. 132-133 ◽

Cited By ~ 1

Author(s):

R. E. Herfert

Keyword(s):

Surface Characterization ◽

Analytical Techniques ◽

X Ray Diffraction ◽

Depth Of Penetration ◽

X Ray ◽

The Past ◽

Transmission Electron ◽

Scanning Electron

Studies of the nature of a surface, either metallic or nonmetallic, in the past, have been limited to the instrumentation available for these measurements. In the past, optical microscopy, replica transmission electron microscopy, electron or X-ray diffraction and optical or X-ray spectroscopy have provided the means of surface characterization. Actually, some of these techniques are not purely surface; the depth of penetration may be a few thousands of an inch. Within the last five years, instrumentation has been made available which now makes it practical for use to study the outer few 100A of layers and characterize it completely from a chemical, physical, and crystallographic standpoint. The scanning electron microscope (SEM) provides a means of viewing the surface of a material in situ to magnifications as high as 250,000X.

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Skeletal elements of crinoid echinoderms: High-resolution structural and microanalytical results

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100074768 ◽

1983 ◽

Vol 41 ◽

pp. 194-195

Author(s):

D. F. Blake ◽

L. F. Allard ◽

D. R. Peacor

Keyword(s):

High Resolution ◽

Marine Invertebrates ◽

Sea Urchins ◽

Structural Features ◽

Sea Stars ◽

High Resolution Tem ◽

Relationship Of ◽

The Relationship ◽

Insight Into

Echinodermata is a phylum of marine invertebrates which has been extant since Cambrian time (c.a. 500 m.y. before the present). Modern examples of echinoderms include sea urchins, sea stars, and sea lilies (crinoids). The endoskeletons of echinoderms are composed of plates or ossicles (Fig. 1) which are with few exceptions, porous, single crystals of high-magnesian calcite. Despite their single crystal nature, fracture surfaces do not exhibit the near-perfect {10.4} cleavage characteristic of inorganic calcite. This paradoxical mix of biogenic and inorganic features has prompted much recent work on echinoderm skeletal crystallography. Furthermore, fossil echinoderm hard parts comprise a volumetrically significant portion of some marine limestones sequences. The ultrastructural and microchemical characterization of modern skeletal material should lend insight into: 1). The nature of the biogenic processes involved, for example, the relationship of Mg heterogeneity to morphological and structural features in modern echinoderm material, and 2). The nature of the diagenetic changes undergone by their ancient, fossilized counterparts. In this study, high resolution TEM (HRTEM), high voltage TEM (HVTEM), and STEM microanalysis are used to characterize tha ultrastructural and microchemical composition of skeletal elements of the modern crinoid Neocrinus blakei.

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Some applications of electron beam microanalysis techniques in VLSI process evaluation

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010007463x ◽

1983 ◽

Vol 41 ◽

pp. 160-161

Author(s):

Simon Thomas

Keyword(s):

Integrated Circuits ◽

Process Evaluation ◽

Large Scale ◽

Technology Development ◽

Analytical Techniques ◽

Successful Implementation ◽

Analysis Of Failures ◽

Characterization Of Materials ◽

Circuits Vlsi

Trends in the technology development of very large scale integrated circuits (VLSI) have been in the direction of higher density of components with smaller dimensions. The scaling down of device dimensions has been not only laterally but also in depth. Such efforts in miniaturization bring with them new developments in materials and processing. Successful implementation of these efforts is, to a large extent, dependent on the proper understanding of the material properties, process technologies and reliability issues, through adequate analytical studies. The analytical instrumentation technology has, fortunately, kept pace with the basic requirements of devices with lateral dimensions in the micron/ submicron range and depths of the order of nonometers. Often, newer analytical techniques have emerged or the more conventional techniques have been adapted to meet the more stringent requirements. As such, a variety of analytical techniques are available today to aid an analyst in the efforts of VLSI process evaluation. Generally such analytical efforts are divided into the characterization of materials, evaluation of processing steps and the analysis of failures.

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Irradiation behavior of pyrolytic silicon carbide

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100074288 ◽

1983 ◽

Vol 41 ◽

pp. 72-73

Author(s):

R. J. Lauf

Keyword(s):

Silicon Carbide ◽

Fission Products ◽

Thermodynamics And Kinetics ◽

Neutron Fluences ◽

Fuel Particles ◽

Sic Coatings ◽

Irradiation Behavior ◽

Kinetics Of ◽

Htgr Fuel

Fuel particles for the High-Temperature Gas-Cooled Reactor (HTGR) contain a layer of pyrolytic silicon carbide to act as a miniature pressure vessel and primary fission product barrier. Optimization of the SiC with respect to fuel performance involves four areas of study: (a) characterization of as-deposited SiC coatings; (b) thermodynamics and kinetics of chemical reactions between SiC and fission products; (c) irradiation behavior of SiC in the absence of fission products; and (d) combined effects of irradiation and fission products. This paper reports the behavior of SiC deposited on inert microspheres and irradiated to fast neutron fluences typical of HTGR fuel at end-of-life.

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Characterization of Tamm-Horsfall Urinary Glycoprotein

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s042482010007254x ◽

1973 ◽

Vol 31 ◽

pp. 420-421

Author(s):

John P. Robinson ◽

J. David Puett

Keyword(s):

Electron Microscopy ◽

Circular Dichroism ◽

Secondary Structure ◽

Quaternary Structure ◽

Normal Adult ◽

Morphological Properties ◽

Adult Males ◽

Circular Dichroïsm ◽

Urinary Glycoprotein

Much work has been reported on the chemical, physical and morphological properties of urinary Tamm-Horsfall glycoprotein (THG). Although it was once reported that cystic fibrotic (CF) individuals had a defective THG, more recent data indicate that THG and CF-THG are similar if not identical.No studies on the conformational aspects have been reported on this glycoprotein using circular dichroism (CD). We examined the secondary structure of THG and derivatives under various conditions and have correlated these results with quaternary structure using electron microscopy.THG was prepared from normal adult males and CF-THG from a 16-year old CF female by the method of Tamm and Horsfall. CF female by the method of Tamm and Horsfall.

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High Resolution Replication of Organoclay Surfaces

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100055126 ◽

1982 ◽

Vol 40 ◽

pp. 576-577

Author(s):

W. W. Barker ◽

W. E. Rigsby ◽

V. J. Hurst ◽

W. J. Humphreys

Keyword(s):

Clay Mineral ◽

Organic Molecule ◽

Organic Molecules ◽

X Ray Diffraction ◽

Xrd Pattern ◽

X Ray ◽

Naturally Occurring ◽

Silicate Layers ◽

Mineral Identification

Experimental clay mineral-organic molecule complexes long have been known and some of them have been extensively studied by X-ray diffraction methods. The organic molecules are adsorbed onto the surfaces of the clay minerals, or intercalated between the silicate layers. Natural organo-clays also are widely recognized but generally have not been well characterized. Widely used techniques for clay mineral identification involve treatment of the sample with H2 O2 or other oxidant to destroy any associated organics. This generally simplifies and intensifies the XRD pattern of the clay residue, but helps little with the characterization of the original organoclay. Adequate techniques for the direct observation of synthetic and naturally occurring organoclays are yet to be developed.

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