Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 419-428 ◽  
Author(s):  
Z. Sheng ◽  
D. Pennica ◽  
W.I. Wood ◽  
K.R. Chien
Keyword(s):  
Cardiac Myocyte ◽  
Embryonic Stem ◽  
Neuronal Cell ◽  
Fold Increase ◽  
Cell System ◽  
Neonatal Rat ◽  
Heart Tube ◽  
Related Family ◽  
Early Expression

We have recently isolated a novel cytokine, cardiotrophin-1 (CT-1), from an in vitro embryonic stem cell system of cardiogenesis that can activate embryonic markers in neonatal rat cardiac myocytes. CT-1 is a new member of the interleukin 6 (IL-6)/leukemia inhibitory factor (LIF) cytokines, which activate downstream signals via gp130-dependent pathways. To define the developmental pattern of expression of CT-1 during murine embryogenesis, we have developed antibodies directed against a CT-1 fusion protein. As assessed by immunolocalization, CT-1 is predominantly expressed in the early mouse embryonic heart tube (E8.5-10.5). In the heart, CT-1 is primarily expressed in myocardial cells, and not in endocardial cushion or outflow tract tissues. After E12.5, CT-1 expression is found in other tissues, including skeletal, liver and dorsal root ganglia. Given the effects of a related family member (ciliary neurotrophic factor, CNTF) on neuronal cell survival, we studied the ability of CT-1 to promote cardiac myocyte survival and proliferation in vitro. Both CT-1 and LIF, which share the same receptors, dramatically promote neonatal cardiac myocyte survival, while IL-6 and CNTF are without effect. A cell proliferation assay documents that CT-1 provokes an approximate 2-fold increase in embryonic cardiac myocyte proliferation. Thus, CT-1 may play an autocrine role during cardiac chamber growth and morphogenesis by promoting the survival and proliferation of immature myocytes, most likely via gp130-dependent signaling pathways.

A novel epithelial cell from neonatal rat lung: isolation and differentiated phenotype

1990 ◽  
Vol 259 (6) ◽  
pp. L415-L425 ◽  
Author(s):  
P. E. Roberts ◽  
D. M. Phillips ◽  
J. P. Mather
Keyword(s):  
Epithelial Cell ◽  
Molecular Mechanisms ◽  
Basal Medium ◽  
Fold Increase ◽  
Cell Types ◽  
Cell Number ◽  
Neonatal Rat ◽  
Rat Lung ◽  
Cell Type

A novel epithelial cell from normal neonatal rat lung has been isolated, established, and maintained for multiple passages in the absence of serum, without undergoing crisis or senescence. By careful manipulation of the nutrition/hormonal microenvironment, we have been able to select, from a heterogeneous population, a single epithelial cell type that can maintain highly differentiated features in vitro. This cell type has characteristics of bronchiolar epithelial cells. A clonal line, RL-65, has been selected and observed for greater than 2 yr in continuous culture. It has been characterized by ultrastructural, morphological, and biochemical criteria. The basal medium for this cell line is Ham's F12/Dulbecco's modified Eagle's (DME) medium plus insulin (1 micrograms/ml), human transferrin (10 micrograms/ml), ethanolamine (10(-4) M), phosphoethanolamine (10(-4) M), selenium (2.5 x 10(-8) M), hydrocortisone (2.5 x 10(-7) M), and forskolin (5 microM). The addition of 150 micrograms/ml of bovine pituitary extract to the defined basal medium stimulates a greater than 10-fold increase in cell number and a 50- to 100-fold increase in thymidine incorporation. The addition of retinoic acid results in further enhancement of cell growth and complete inhibition of keratinization. We have demonstrated a strategy that may be applicable to isolating other cell types from the lung and maintaining their differentiated characteristics for long-term culture in vitro. Such a culture system promises to be a useful model in which to study cellular events associated with differentiation and proliferation in the lung and to better understand the molecular mechanisms involved in these events.

Coverslip hypoxia: a novel method for studying cardiac myocyte hypoxia and ischemia in vitro

2004 ◽  
Vol 287 (4) ◽  
pp. H1801-H1812 ◽  
Author(s):  
Kelly R. Pitts ◽  
Christopher F. Toombs
Keyword(s):  
Metabolic Activity ◽  
Cardiac Myocyte ◽  
Experimental Models ◽  
Membrane Dynamics ◽  
Neonatal Rat ◽  
Simple Method ◽  
Glass Coverslip ◽  
Media Volume

In vitro experimental models designed to study the effects of hypoxia and ischemia typically employ oxygen-depleted media and/or hypoxic chambers. These approaches, however, allow for metabolites to diffuse away into a large volume and may not replicate the high local concentrations that occur in ischemic myocardium in vivo. We describe herein a novel and simple method for creating regional hypoxic and ischemic conditions in neonatal rat cardiac myocyte monolayers. This method consists of creating a localized diffusion barrier by placing a glass coverslip over a portion of the monolayer. The coverslip restricts covered myocytes to a thin film of media while leaving uncovered myocytes free to access the surrounding bulk media volume. Myocytes under the coverslip undergo marked morphology changes over time as assessed by video microscopy. Fluorescence microscopy shows that these changes are accompanied by alterations in mitochondrial membrane potential and plasma membrane dynamics and eventually result in myocyte death. We also show that the metabolic activity of myocytes drives cell necrosis under the coverslip. In addition, the intracellular pH of synchronously contracting myocytes under the coverslip drops rapidly, which further implicates metabolic activity in regulating cell death under the coverslip. In contrast with existing models of hypoxia/ischemia, this technique provides a simple and effective way to create hypoxic/ischemic conditions in vitro. Moreover, we conclude that myocyte death is hastened by the combination of hypoxia, metabolites, and acidosis and is facilitated by a reduction in media volume, which may better represent ischemic conditions in vivo.

scholarly journals Simple Method to Isolation and Culture of Neuron Progenitor Cells (NPCs) from Whole Brain Post-Natal Rat

2018 ◽  
Vol 9 (2) ◽  
pp. 63-69
Author(s):  
ARIYANI NOVIANTARI ◽  
Masagus Zainuri ◽  
Ratih Rinendyaputri ◽  
Ni Ketut Susilarini
Keyword(s):  
Progenitor Cells ◽  
Neuronal Cell ◽  
Neonatal Rat ◽  
Sprague Dawley ◽  
Simple Method ◽  
Whole Brain ◽  
Culture Cells ◽  
Result Show ◽  
Neuronal Cell Lines

Background: Using of neuron cells for in vitro neurobiology study is needed. Neuron cell can be obtained from a primary neuron or neuronal cell lines, depend on the aim of the study because both are not equivalent. Various methods are performed to obtain primary neurons from the cortical, hippocampal and whole brain of pre or neonatal rat. The limitations of neuron cells to proliferate so that is necessary to develop a method to isolate neuron progenitor cells (NPCs). The aim of the present study was to isolate NPCs from whole brain post-natal rat.   Methods: Whole brain were obtained from neonates Sprague Dawley rat. There are 2 step to get NSC; first isolation by taking the brain into the 15 ml of tube with 1 ml of  0,05% trypsin EDTA for 400g brain (incubated in the 370C, 5% CO2 for 10 minutes),  tirturation with adding 1 ml culture medium  and 5 ml HBSS-glucose then filtered by 70μm pore size membrane and centrifuged  2000 rpm for 10 minutes. Second: remove of supernatant with add 1 ml of HBSS-Glucose and taking it into a tube with  35% and 65% concentration of Ficoll then centrifuged at 1800 g for 10 minutes then supernatant were replated twice with poly D lysine (100µg/ml). Characterization of progenitor neuron immunotype was checked by immunohistochemistry with positive marker (NeuN and MAP2) and flow cytometry (PSANCAM+ and A2B5 -). Results: In this study, our result show that this method does not take longer than one hours and > 95% cells that obtained are expressing PSANCAM+.  After 4 days culture, cells exhibit positive for neuron marker as MAP2 and NeuN.   Conclusion: The method that our develope to isolate neuron progenitor cell from whole-brain are more effective and more simple with high viability and purity.

Abstract P379: Inhibition Of O-glcnac Transferase Ogt Activates P38 Signaling In Neonatal Rat Cardiomyocytes

2021 ◽  
Vol 129 (Suppl_1) ◽  
Author(s):  
Kyriakos Papanicolaou ◽  
Natasha Zachara ◽  
Deepthi Ashok ◽  
Agnes Sidor ◽  
D B Foster ◽  
...  
Keyword(s):  
Heart Failure ◽  
Intracellular Signaling ◽  
Ventricular Myocytes ◽  
Fold Increase ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Rat Cardiomyocytes ◽  
Creb Phosphorylation ◽  
Glcnac Transferase

The mitogen activated protein kinase (MAPK) p38 is important in cardiac hypertrophic responses and p38 inhibition has been tested as a potential therapeutic approach to heart failure. p38 is tightly regulated by upstream kinases and phosphatases. While p38 inhibitors suppress cardiac hypertrophy in vitro and in animal models, the partial efficacy of p38 inhibitors in clinical trials for heart failure illustrates the need for a deeper understanding of p38-regulatory mechanisms. O -linked N-Acetylglucosamine ( O -GlcNAc) on Ser/Thr residues is a ubiquitous intracellular modification ( O -GlcNAcylation) that participates in intracellular signaling, often occurring in counterpoint to phosphorylation. O -GlcNAcylation is catalyzed by O -GlcNAc Transferase (OGT) and removed by O -GlcNAc-Ase (OGA). Given the crucial regulation of p38 activity by phosphorylation, we hypothesized that O -GlcNAcylation regulates p38 phosphorylation during basal and hypertrophic cardiomyocyte signaling. Treating neonatal rat ventricular myocytes (NRVM) with OSMI-1 (inhibitor of OGT) significantly decreased O -GlcNAcylation (0.48 ± 0.02, P <0.001 vs. vehicle), whereas treatment with Thiamet-G (inhibitor of OGA) significantly increased O -GlcNAcylation (3.0-fold increase ± 0.35, P <0.05 vs. vehicle). OSMI1 treatment induced the phosphorylation of p38 at its activation site (3.9-fold increase ± 0.46, P <0.001 vs. vehicle) and promoted the phosphorylation of the downstream target, heat shock protein Hsp27 (8-fold increase ± 1.3, P <0.0001 vs. vehicle) and transcription factor Creb (3.3-fold increase ± 0.12, P <0.001 vs. vehicle). OSMI-1 had an additive effect in inducing p38 and Creb phosphorylation following hypertrophic stimulation by phenylephrine (3.1-fold and 1.4-fold increase vs. phenylephrine respectively, P <0.05). Treatment with the p38 inhibitor SB202190 abolished the phosphorylation of Hsp27 and Creb that was induced by OSMI-1. Canonical upstream activators of p38 include the MAP3Ks, TAK1 and ASK1. However, we found that treatment with ASK1 or TAK1 inhibitors (GS-444217 and Takinib, respectively) either alone, or in combination, did not negate the phosphorylation of p38 by OSMI-1. We conclude that regulation of p38 by OGT activity could occur at a level downstream of canonical MAP3Ks or through non-canonical pathways.

scholarly journals Development of electrical activity in cardiac myocyte aggregates derived from mouse embryonic stem cells

2003 ◽  
Vol 284 (6) ◽  
pp. H2114-H2123 ◽  
Author(s):  
K. Banach ◽  
M. D. Halbach ◽  
P. Hu ◽  
J. Hescheler ◽  
U. Egert
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Action Potential ◽  
Electrical Activity ◽  
Cardiac Myocyte ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Electrophysiological Properties ◽  
Chronotropic Response

Embryonic stem cells differentiate into cardiac myocytes, repeating in vitro the structural and molecular changes associated with cardiac development. Currently, it is not clear whether the electrophysiological properties of the multicellular cardiac structure follow cardiac maturation as well. In long-term recordings of extracellular field potentials with microelectrode arrays consisting of 60 substrate-integrated electrodes, we examined the electrophysiological properties during the ongoing differentiation process. The beating frequency of the growing preparations increased from 1 to 5 Hz concomitant to a decrease of the action potential duration and action potential rise time. A developmental increase of the conduction velocity could be attributed to an increased expression of connexin43 gap junction channels. Whereas isoprenalin elicited a positive chronotropic response from the first day of spontaneous beating onward, a concentration-dependent negative chronotropic effect of carbachol only developed after ∼4 days. The in vitro development of the three-dimensional cardiac preparation thus closely follows the development described for the mouse embryonic heart, making it an ideal model to monitor the differentiation of electrical activity in embryonic cardiomyocytes.

scholarly journals Species-specific pace of development is associated with differences in protein stability

Science ◽  
2020 ◽  
Vol 369 (6510) ◽  
pp. eaba7667 ◽  
Author(s):  
Teresa Rayon ◽  
Despina Stamataki ◽  
Ruben Perez-Carrasco ◽  
Lorena Garcia-Perez ◽  
Christopher Barrington ◽  
...  
Keyword(s):  
Protein Stability ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Genomic Sequence ◽  
Embryonic Stem ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Cycle Duration ◽  
Species Specific

Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which the embryo develops can vary substantially between species. For example, the same genetic program, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the program runs more than twice as fast in mouse as in human. This is not due to differences in signaling, nor the genomic sequence of genes or their regulatory elements. Instead, there is an approximately two-fold increase in protein stability and cell cycle duration in human cells compared with mouse cells. This can account for the slower pace of human development and suggests that differences in protein turnover play a role in interspecies differences in developmental tempo.

scholarly journals Use of human embryonic stem cells to model pediatric gliomas with H3.3K27M histone mutation

Science ◽  
2014 ◽  
Vol 346 (6216) ◽  
pp. 1529-1533 ◽  
Author(s):  
Kosuke Funato ◽  
Tamara Major ◽  
Peter W. Lewis ◽  
C. David Allis ◽  
Viviane Tabar
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Cell System ◽  
Genome Wide ◽  
Regulator Genes

Over 70% of diffuse intrinsic pediatric gliomas, an aggressive brainstem tumor, harbor heterozygous mutations that create a K27M amino acid substitution (methionine replaces lysine 27) in the tail of histone H3.3. The role of the H3.3K27M mutation in tumorigenesis is not fully understood. Here, we use a human embryonic stem cell system to model this tumor. We show that H3.3K27M expression synergizes with p53 loss and PDGFRA activation in neural progenitor cells derived from human embryonic stem cells, resulting in neoplastic transformation. Genome-wide analyses indicate a resetting of the transformed precursors to a developmentally more primitive stem cell state, with evidence of major modifications of histone marks at several master regulator genes. Drug screening assays identified a compound targeting the protein menin as an inhibitor of tumor cell growth in vitro and in mice.

scholarly journals Differential Expression of α, β, and γ Protocadherin Isoforms During Differentiation, Aging, and Cancer

2021 ◽  
Author(s):  
Michael D. West ◽  
Ivan Labat ◽  
Jie Li ◽  
Pam Sim ◽  
Jeffrey Janus ◽  
...  
Keyword(s):  
Potential Role ◽  
Replicative Senescence ◽  
Cpg Island ◽  
Embryonic Stem ◽  
Neuronal Cell ◽  
Differential Regulation ◽  
Ctcf Binding ◽  
Cell Recognition ◽  
Cell Cell

AbstractThe cadherin family of cell surface glycoproteins plays a fundamental role in cell-cell recognition, thereby participating in diverse biological process such as embryonic morphogenesis and oncogenic transformation. The subset of clustered protocadherin (PCDH) genes generated from the α, β, and γ loci, have been widely studied for their potential role in neuronal cell-cell recognition and neurogenesis, however their broader role in normal embryonic development and cancer has not been examined in detail. We utilized human embryonic stem (hES) cells to model early human developmentin vitro, comparing PCDH isoform transcription in diverse types of embryonic progenitors with normal adult-derived and cancer counterparts. Embryonic progenitors express genes from the α and β cluster at levels comparable to that seen in the CNS, while fetal and adult-derived cells express primarily from the γ cluster. Replicative senescence left fibroblasts with markedly lower expression of all isoforms. We observe that an embryonic pattern of clustered protocadherin gene expression and associated CpG island methylation is commonly associated with cancer cell lines from diverse tissue types. The differential regulation of the α, β, and γ loci coincide with alternate regions of DNA accessibility at CTCF binding sites and lamina-associated domains and CPL expression correlated with the expression ofLMNAandLMNB1. These observations support a potential role for the differential regulation of genes within the clustered protocadherin locus in selective cell-cell adhesion during embryogenesis, regeneration, cancer and aging.

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