Conditional CRISPR‐mediated deletion of Lyn kinase enhances differentiation and function of iPSC‐derived megakaryocytes

2021 ◽  
Author(s):  
Alyssa J. Moroi ◽  
Peter J. Newman
Keyword(s):  
Lyn Kinase ◽  
And Function

scholarly journals Multiple roles of Lyn kinase in myeloid cell signaling and function

2009 ◽  
Vol 228 (1) ◽  
pp. 23-40 ◽  
Author(s):  
Patrizia Scapini ◽  
Shalini Pereira ◽  
Hong Zhang ◽  
Clifford A. Lowell
Keyword(s):  
Cell Signaling ◽  
Myeloid Cell ◽  
Multiple Roles ◽  
Lyn Kinase ◽  
And Function

scholarly journals Conditional CRISPR-Mediated Deletion of Lyn Kinase Promotes Megakaryocyte Differentiation, Platelet Production and Function

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 30-30
Author(s):  
Alyssa J. Moroi ◽  
Peter J. Newman
Keyword(s):  
Stem Cells ◽  
Gene Editing ◽  
Negative Regulator ◽  
Differentiation Process ◽  
Ips Cell ◽  
Platelet Production ◽  
Lyn Kinase ◽  
And Function ◽  
Megakaryocyte Differentiation

Thrombocytopenia leading to life-threatening excessive bleeding can be caused by defects in megakaryocyte/platelet production, platelet depletion by platelet-specific auto- or allo-antibodies, blood loss following trauma or surgery, or as a result of damage to bone marrow blood stem cells induced by chemo- or radiation therapy. Currently, platelets available for transfusion are entirely dependent on volunteer blood donation, which are often in short supply, carry a risk of infection, and often unable to be antigen-matched for alloimmunized patients. Thus, maintaining an adequate supply of platelets for transfusion is often a challenge. In response to this growing clinical need, a number of laboratories have started differentiating human induced pluripotent stem cells (iPSCs) into megakaryocytes, with the long-term goal of creating a readily available supply of antigen-matched, highly functional platelets. Current methods for generating platelets from iPS cell-derived megakaryocytes have been improved to produce enough platelets in vitro to begin phase I safety trials in humans; however, modifying the iPS cell genome has the potential to further improve both the yield and function of in vitro-generated platelets. The purpose of this investigation, therefore, was to combine recent advances in gene editing technology with iPS cell differentiation techniques to improve the yield and/or functionality of in vitro-derived platelet products. As a start, we sought to examine whether disruption of negative regulators of platelet production and/or activation might allow the generation of increased numbers of platelets with improved hemostatic effectiveness. One such negative regulator of platelet function is the Src family kinase, Lyn. Previous studies using pharmacologic inhibitors and global Lyn knockout mice have shown that that disrupting Lyn activity leads to mild thrombocytosis and megakaryocytosis. The effects of specifically targeting Lyn in human megakaryocytes and platelets, however, are not known. We hypothesized that conditionally disrupting Lyn during the process of iPSC to megakaryocyte differentiation might be an effective way to enhance megakaryocyte differentiation, platelet production, and hemostatic function. Using CRISPR/Cas9 gene editing technology, we generated a temporally controllable, tamoxifen-inducible Lyn-KO iPS cell line system that allowed us to conditionally delete Lyn at any desired stage of the iPSC→hematopoietic stem cell (HSC)→megakaryocyte→platelet differentiation process. We found tamoxifen efficiently disrupted Lyn expression at any of these stages within 24-48 hours after addition of the drug to the cell culture, and also improved the efficiency and yield of the iPSC→megakaryocyte→proplatelet differentiation process. Lyn-deficient human megakaryocytes prepared in this fashion exhibited enhanced reactivity, as reported by increased Erk phosphorylation in response to thrombopoietin stimulation, and by an improved ability to support hemostasis in human blood in vitro, as determined using thromboelastometry (ROTEM). Taken together, these data suggest that temporal disruption of Lyn kinase might represent an effective strategy for the engineering and manufacture of in vitro-derived platelets for both diagnostic and future therapeutic use. Disclosures Newman: NIH: Research Funding; Bloodworks Northwest: Membership on an entity's Board of Directors or advisory committees; Rallybio Corporation: Consultancy.

scholarly journals Lyn kinase promotes erythroblast expansion and late-stage development

Blood ◽  
2006 ◽  
Vol 108 (5) ◽  
pp. 1524-1532 ◽  
Author(s):  
Vinit G. Karur ◽  
Clifford A. Lowell ◽  
Peter Besmer ◽  
Valter Agosti ◽  
Don M. Wojchowski
Keyword(s):  
Late Stage ◽  
Progenitor Cell ◽  
Ex Vivo ◽  
Cell Formation ◽  
Erythroid Cell ◽  
Cycle Phase ◽  
Lyn Kinase ◽  
Death Associated Protein Kinase ◽  
Stage Development ◽  
And Function

Lyn kinase is known to modulate the formation and function of B cells, monocytes, and mast cells. However, Lyn-/- mice also develop erythrosplenomegaly, and cases for both negative and positive erythropoietic actions of Lyn recently have been outlined. In phenylhydrazine-treated Lyn-/- mice, extramedullary splenic erythropoiesis was hyperactivated, but this did not lead to accelerated recovery from anemia. Furthermore, ex vivo analyses of the development of bone marrow-derived Lyn-/- erythroblasts in unique primary culture systems indicated positive roles for Lyn at 2 stages. Late-stage Lyn-/- erythroblasts exhibited deficit Ter119pos cell formation, and this was paralleled by increased apoptosis (and decreased Bcl-xL expression). During early development, Lyn-/- erythroblasts accumulated at a KitposCD71high stage, possessed decreased proliferative capacity, and were attenuated in entering an apparent G1/S cell-cycle phase. In proposed compensatory responses, Lyn-/- erythroblasts expressed increased levels of activated Akt and p60-Src and decreased levels of death-associated protein kinase-2. Stat5 activation and Bcl-xL expression, in contrast, were significantly decreased in keeping with decreased survival and developmental potentials. Lyn, therefore, is proposed to function via erythroid cell-intrinsic mechanisms to promote progenitor cell expansion beyond a KitposCD71high stage and to support subsequent late-stage development.

Conformation of Ribosomes from Escherichia Coli

1974 ◽  
Vol 32 ◽  
pp. 210-211
Author(s):  
M. Boublik ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Binding Sites ◽  
Ribosomal Proteins ◽  
Fluorescent Dyes ◽  
Protein Biosynthesis ◽  
Three Dimensional ◽  
Spatial Arrangement ◽  
Dimensional Structure ◽  
Complete Understanding ◽  
And Function

The present knowledge of the three-dimensional structure of ribosomes is far too limited to enable a complete understanding of the various roles which ribosomes play in protein biosynthesis. The spatial arrangement of proteins and ribonuclec acids in ribosomes can be analysed in many ways. Determination of binding sites for individual proteins on ribonuclec acid and locations of the mutual positions of proteins on the ribosome using labeling with fluorescent dyes, cross-linking reagents, neutron-diffraction or antibodies against ribosomal proteins seem to be most successful approaches. Structure and function of ribosomes can be correlated be depleting the complete ribosomes of some proteins to the functionally inactive core and by subsequent partial reconstitution in order to regain active ribosomal particles.

The Effect of Oxidized Cholesterol on the Aorta of Rabbits- Scanning Electron Microscopic Observations

1982 ◽  
Vol 40 ◽  
pp. 340-341
Author(s):  
S. K. Pena ◽  
C. B. Taylor ◽  
J. Hill ◽  
J. Safarik
Keyword(s):  
Cholesterol Biosynthesis ◽  
Cholesterol Content ◽  
Arterial Injury ◽  
Electron Microscopic ◽  
Aortic Smooth Muscle ◽  
Oxidized Cholesterol ◽  
Initial Event ◽  
Membrane Structure And Function ◽  
Scanning Electron Microscopic ◽  
And Function

Introduction: Oxidized cholesterol derivatives have been demonstrated in various cell cultures to be very potent inhibitors of 3-hvdroxy-3- methylglutaryl Coenzyme A reductase which is a principle regulator of cholesterol biosynthesis in the cell. The cholesterol content in the cells exposed to oxidized cholesterol was found to be markedly decreased. In aortic smooth muscle cells, the potency of this effect was closely related to the cytotoxicity of each derivative. Furthermore, due to the similarity of their molecular structure to that of cholesterol, these oxidized cholesterol derivatives might insert themselves into the cell membrane, alter membrane structure and function and eventually cause cell death. Arterial injury has been shown to be the initial event of atherosclerosis.

Ultrastructure of developing visual cortical synapses in the cat superior colliculus

1991 ◽  
Vol 49 ◽  
pp. 156-157
Author(s):  
Caroline A. Miller ◽  
Laura L. Bruce
Keyword(s):  
Superior Colliculus ◽  
Phosphate Buffer ◽  
Receptive Fields ◽  
Visual Cortical Area ◽  
Cortical Synapses ◽  
Visual Cortical ◽  
And Function ◽  
Phosphate Buffered Saline ◽  
The Brain ◽  
Cat Superior Colliculus

The first visual cortical axons arrive in the cat superior colliculus by the time of birth. Adultlike receptive fields develop slowly over several weeks following birth. The developing cortical axons go through a sequence of changes before acquiring their adultlike morphology and function. To determine how these axons interact with neurons in the colliculus, cortico-collicular axons were labeled with biocytin (an anterograde neuronal tracer) and studied with electron microscopy.Deeply anesthetized animals received 200-500 nl injections of biocytin (Sigma; 5% in phosphate buffer) in the lateral suprasylvian visual cortical area. After a 24 hr survival time, the animals were deeply anesthetized and perfused with 0.9% phosphate buffered saline followed by fixation with a solution of 1.25% glutaraldehyde and 1.0% paraformaldehyde in 0.1M phosphate buffer. The brain was sectioned transversely on a vibratome at 50 μm. The tissue was processed immediately to visualize the biocytin.

Structural modifications of intercellular junctions.

1978 ◽  
Vol 36 (2) ◽  
pp. 330-331
Author(s):  
J. Metz ◽  
M. Merlo ◽  
W. G. Forssmann
Keyword(s):  
Gap Junctions ◽  
Intercellular Junctions ◽  
Cell Isolation ◽  
Extrahepatic Cholestasis ◽  
Structural Modifications ◽  
Pancreatic Duct Ligation ◽  
Pancreatic Cells ◽  
Duct Ligation ◽  
Thin Sectioning ◽  
And Function

Structure and function of intercellular junctions were studied under the electronmicroscope using conventional thin sectioning and freeze-etch replicas. Alterations of tight and gap junctions were analyzed 1. of exocrine pancreatic cells under cell isolation conditions and pancreatic duct ligation and 2. of hepatocytes during extrahepatic cholestasis.During the different steps of cell isolation of exocrine pancreatic cells, gradual changes of tight and gap junctions were observed. Tight junctions, which formed belt-like structures around the apex of control acinar cells in situ, subsequently diminished, became interrupted and were concentrated into macular areas (Fig. 1). Aggregations of membrane associated particles, which looked similar to gap junctions, were intermixed within tight junctional areas (Fig. 1). These structures continously disappeared in the last stages of the isolation procedure. The intercellular junctions were finally separated without destroying the integrity of the cell membrane, which was confirmed with porcion yellow, lanthanum chloride and horse radish peroxidase.

Structural similarity of ribosomes from E. coli and A. salina

1978 ◽  
Vol 36 (2) ◽  
pp. 242-243
Author(s):  
M. Boublik ◽  
R.M. Wydro ◽  
W. Hellmann ◽  
F. Jenkins
Keyword(s):  
Ribosomal Proteins ◽  
Direct Evidence ◽  
Structural Similarity ◽  
Carbon Support ◽  
Artemia Salina ◽  
Uranyl Acetate ◽  
Biological Assays ◽  
E Coli ◽  
And Function ◽  
Fine Carbon

Ribosomes are ribonucleoprotein particles necessary for processing the genetic information of mRNA into proteins. Analogy in composition and function of ribosomes from diverse species, established by biochemical and biological assays, implies their structural similarity. Direct evidence obtained by electron microscopy seems to be of increasing relevance in understanding the structure of ribosomes and the mechanism of their role in protein synthesis.The extent of the structural homology between prokaryotic and eukaryotic ribosomes has been studied on ribosomes of Escherichia coli (E.c.) and Artemia salina (A.s.). Despite the established differences in size and in the amount and proportion of ribosomal proteins and RNAs both types of ribosomes show an overall similarity. The monosomes (stained with 0.5% aqueous uranyl acetate and deposited on a fine carbon support) appear in the electron micrographs as round particles with a diameter of approximately 225Å for the 70S E.c. (Fig. 1) and 260Å for the 80S A.s. monosome (Fig. 2).

The Scanning Electron Microscopic Observation of the Vestibular Sensory Epithelia

1969 ◽  
Vol 27 ◽  
pp. 40-41
Author(s):  
D.J. Lim ◽  
W.C. Lane
Keyword(s):  
Semicircular Canals ◽  
Electron Microscopic Observation ◽  
Gravitational Acceleration ◽  
Electron Microscopic ◽  
Sensory Epithelia ◽  
Scanning Electron Microscopic ◽  
Vestibular Sensory Epithelia ◽  
And Function ◽  
Sand Piles ◽  
Active Investigation

The morphology and function of the vestibular sensory organs has been extensively studied during the last decade with the advent of electron microscopy and electrophysiology. The opening of the space age also accelerated active investigation in this area, since this organ is responsible for the sensation of balance and of linear, angular and gravitational acceleration.The vestibular sense organs are formed by the saccule, utricle and three ampullae of the semicircular canals. The maculae (sacculi and utriculi) have otolithic membranes on the top of the sensory epithelia. The otolithic membrane is formed by a layer of thick gelatin and sand-piles of calcium carbonate crystals (Fig.l).

Structure and function of neural networks in the retina

1988 ◽  
Vol 46 ◽  
pp. 26-27
Author(s):  
Peter Sterling
Keyword(s):  
Ganglion Cells ◽  
Three Dimensional ◽  
Structure And Function ◽  
Synaptic Connections ◽  
Visual Axis ◽  
One Dimensional ◽  
Cat Retina ◽  
Ultrathin Sections ◽  
And Function ◽  
Insight Into

The synaptic connections in cat retina that link photoreceptors to ganglion cells have been analyzed quantitatively. Our approach has been to prepare serial, ultrathin sections and photograph en montage at low magnification (˜2000X) in the electron microscope. Six series, 100-300 sections long, have been prepared over the last decade. They derive from different cats but always from the same region of retina, about one degree from the center of the visual axis. The material has been analyzed by reconstructing adjacent neurons in each array and then identifying systematically the synaptic connections between arrays. Most reconstructions were done manually by tracing the outlines of processes in successive sections onto acetate sheets aligned on a cartoonist's jig. The tracings were then digitized, stacked by computer, and printed with the hidden lines removed. The results have provided rather than the usual one-dimensional account of pathways, a three-dimensional account of circuits. From this has emerged insight into the functional architecture.

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