scholarly journals High-throughput micro-patterning platform reveals Nodal-dependent dissection of peri-gastrulation-associated versus pre-neurulation associated fate patterning

2018 ◽  
Author(s):  
Mukul Tewary ◽  
Dominika Dziedzicka ◽  
Joel Ostblom ◽  
Laura Prochazka ◽  
Nika Shakiba ◽  
...  
Keyword(s):  
Reaction Diffusion ◽  
Positional Information ◽  
Significant Heterogeneity ◽  
Gene Profile ◽  
Self Organized ◽  
Micro Patterning ◽  
Post Implantation ◽  
Geometrically Confined

AbstractIn vitro models of post-implantation human development are valuable to the fields of regenerative medicine and developmental biology. Here, we report characterization of a robust in vitro platform that enabled high-content screening of multiple human pluripotent stem cell (hPSC) lines for their ability to undergo peri-gastrulation-like fate patterning upon BMP4 treatment of geometrically-confined colonies and observed significant heterogeneity in their differentiation propensities along a gastrulation associable and neuralization associable axis. This cell line associated heterogeneity was found to be attributable to endogenous nodal expression, with upregulation of Nodal correlated with expression of a gastrulation-associated gene profile, and Nodal downregulation correlated with a neurulation-associated gene profile expression. We harness this knowledge to establish a platform of pre-neurulation-like fate patterning in geometrically confined hPSC colonies that arises due to a stepwise activation of reaction-diffusion and positional-information. Our work identifies a Nodal signalling dependent switch in peri-gastrulation versus pre-neurulation-associated fate patterning in hPSC cells, provides a technology to robustly assay hPSC differentiation outcomes, and suggests conserved mechanisms of self-organized fate specification in differentiating epiblast and ectodermal tissues.

Characterization of human blood dendritic cell subsets

Blood ◽  
2002 ◽  
Vol 100 (13) ◽  
pp. 4512-4520 ◽  
Author(s):  
Kelli P. A. MacDonald ◽  
David J. Munster ◽  
Georgina J. Clark ◽  
Andrzej Dzionek ◽  
Juergen Schmitz ◽  
...  
Keyword(s):  
Clinical Settings ◽  
Significant Heterogeneity ◽  
Differentiation Antigen ◽  
Peripheral Blood Mononuclear ◽  
Hla Dr ◽  
The Individual ◽  
Antigen Presenting ◽  
Blood Dendritic Cell

Dendritic cells (DCs) are key antigen-presenting cells for stimulating immune responses and they are now being investigated in clinical settings. Although defined as lineage-negative (Lin−) HLA-DR+ cells, significant heterogeneity in these preparations is apparent, particularly in regard to the inclusion or exclusion of CD14+, CD16+, and CD2+ cells. This study used flow cytometry and a panel of monoclonal antibodies (mAbs), including reagents from the 7th Leukocyte Differentiation Antigen Workshop, to define the cellular composition of 2 standardized peripheral blood mononuclear cell (PBMCs)–derived Lin− HLA-DR+preparations. Lin− cells were prepared from PBMCs by depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs. Analysis of the CD16-replete preparations divided the Lin− HLA-DR+ population into 5 nonoverlapping subsets (mean ± 1 SD): CD123 (mean = 18.3% ± 9.7%), CD1b/c (18.6% ± 7.6%), CD16 (49.6% ± 8.5%), BDCA-3 (2.7% ± 1.4%), and CD34 (5.0% ± 2.4%). The 5 subsets had distinct phenotypes when compared with each other, monocytes, and monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were also expressed differentially on the 5 Lin−HLA-DR+ subsets, monocytes, and MoDCs. The poor viability of CD123+ DCs in vitro was confirmed, but the CD16+ CD11c+ DC subset also survived poorly. Finally, the individual subsets used as stimulators in allogeneic mixed leukocyte reactions were ranked by their allostimulatory capacity as CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide an opportunity to standardize the DC populations used for future molecular, functional and possibly even therapeutic studies.

scholarly journals A stepwise model of reaction-diffusion and positional information governs self-organized human peri-gastrulation-like patterning

Development ◽  
2017 ◽  
Vol 144 (23) ◽  
pp. 4298-4312 ◽  
Author(s):  
Mukul Tewary ◽  
Joel Ostblom ◽  
Laura Prochazka ◽  
Teresa Zulueta-Coarasa ◽  
Nika Shakiba ◽  
...  
Keyword(s):  
Reaction Diffusion ◽  
Positional Information ◽  
Self Organized

scholarly journals Spatio-temporal patterning of living cells with extracellular DNA programs

2020 ◽  
Author(s):  
Marc Van Der Hofstadt ◽  
Jean-Christophe Galas ◽  
André Estevez-Torres
Keyword(s):  
Reaction Diffusion ◽  
Positional Information ◽  
Living Cells ◽  
Extracellular Dna ◽  
Extracellular Medium ◽  
Equilibrium Dynamics ◽  
Cell Internalization ◽  
Spatio Temporal ◽  
Non Equilibrium

AbstractReactive extracellular media focus on engineering reaction networks outside the cell to control intracellular chemical composition across time and space. However, current implementations lack the feedback loops and out-of-equilibrium molecular dynamics for encoding spatio-temporal control. Here, we demonstrate that enzyme-DNA molecular programs combining these qualities are functional in an extracellular medium where human cells can grow. With this approach, we construct an internalization program that delivers fluorescent DNA inside living cells and remains functional for at least 48 h. Its non-equilibrium dynamics allows us to control both the time and position of cell internalization. In particular, a spatially inhomogeneous version of this program generates a tunable reaction-diffusion two-band pattern of cell internalization. This demonstrates that a synthetic extracellular program can provide temporal and positional information to living cells, emulating archetypal mechanisms observed during embryo development. We foresee that non-equilibrium reactive extracellular media could be advantageously applied to in vitro biomolecular tracking, tissue engineering or smart bandages.

Assays for Phospholipid-Dependent Formation of Thrombin and Xa: A Potential Method for Quantifying Lupus Anticoagulant Activity

1991 ◽  
Vol 66 (04) ◽  
pp. 453-458 ◽  
Author(s):  
John T Brandt
Keyword(s):  
Specific Activity ◽  
Anticoagulant Activity ◽  
Factor Xa ◽  
Clinical Importance ◽  
Potential Method ◽  
Quantitative Expression ◽  
Increased Risk ◽  
Apparent Association

SummaryLupus anticoagulants (LAs) are antibodies which interfere with phospholipid-dependent procoagulant reactions. Their clinical importance is due to their apparent association with an increased risk of thrombo-embolic disease. To date there have been few assays for quantifying the specific activity of these antibodies in vitro and this has hampered attempts to purify and characterize these antibodies. Methods for determining phospholipid-dependent generation of thrombin and factor Xa are described. Isolated IgG fractions from 7 of 9 patients with LAs were found to reproducibly inhibit enzyme generation in these assay systems, permitting quantitative expression of inhibitor activity. Different patterns of inhibitory activity, based on the relative inhibition of thrombin and factor Xa generation, were found, further substantiating the known heterogeneity of these antibodies. These systems may prove helpful in further purification and characterization of LAs.

Characterization of the Original Christmas Disease Mutation (Cysteine 206 → Serine): From Clinical Recognition to Molecular Pathogenesis

1992 ◽  
Vol 67 (01) ◽  
pp. 063-065 ◽  
Author(s):  
Sherryl A M Taylor ◽  
Jacalyn Duffin ◽  
Cherie Cameron ◽  
Jerome Teitel ◽  
Bernadette Garvey ◽  
...  
Keyword(s):  
Nucleotide Substitution ◽  
Novel Mutation ◽  
Factor Ix ◽  
Causative Mutation ◽  
Coding Region ◽  
Body Of Knowledge ◽  
Polymerase Chain ◽  
Splice Junctions

SummaryChristmas disease was first reported as a distinct clinical entity in two manuscripts published in 1952 (1, 2). The eponym associated with this disorder, is the surname of the first patient examined in detail and reported by Biggs and colleagues in a paper describing the clinical and laboratory features of seven affected individuals (3). This patient has severe factor IX coagulant deficiency (less than 0.01 units/ml) and no detectable circulating factor IX antigen (less than 0.01 units/ml). Coding sequence and splice junctions of the factor IX gene from this patient have been amplified in vitro through the polymerase chain reaction (PCR). One nucleotide substitution was identified at nucleotide 30,070 where a guanine was replaced by a cytosine. This mutation alters the amino acid encoded at position 206 in the factor IX protein from cysteine to serine. The non conservative nature of this substitution, the absence of this change in more than 200 previously sequenced factor IX genes and the fact that the remainder of the coding region of this gene was normal, all provide strong circumstantial evidence in favour of this change being the causative mutation in this patient. The molecular characterization of this novel mutation in the index case of Christmas disease, contributes to the rapidly expanding body of knowledge pertaining to Christmas disease pathogenesis.

IN VITRO/IN SILICO CHARACTERIZATION OF ACTIVE AND PASSIVE STRESSES IN CARDIAC MUSCLE

2012 ◽  
Vol 10 (2) ◽  
pp. 171-188 ◽  
Author(s):  
Markus Boel ◽  
Oscar J. Abilez ◽  
Ahmed N Assar ◽  
Christopher K. Zarins ◽  
Ellen Kuhl
Keyword(s):  
Cardiac Muscle ◽  
In Silico ◽  
In Silico Characterization
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