scholarly journals Mouse embryonic stem cell-derived cardiomyocytes cease to beat following exposure to monochromatic light: association with increased ROS and loss of calcium transients

2019 ◽  
Vol 317 (4) ◽  
pp. C725-C736
Author(s):  
Gurbind Singh ◽  
Divya Sridharan ◽  
Mahmood Khan ◽  
Polani B. Seshagiri
Keyword(s):  
Cell Biology ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Monochromatic Light ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Medical Diagnostics ◽  
Calcium Transients ◽  
Egfp Expression ◽  
Mitochondrial Activity

We earlier established the mouse embryonic stem (ES) cell “GS-2” line expressing enhanced green fluorescent protein (EGFP) and have been routinely using it to understand the molecular regulation of differentiation into cardiomyocytes. During such studies, we made a serendipitous discovery that functional cardiomyocytes derived from ES cells stopped beating when exposed to blue light. We observed a gradual cessation of contractility within a few minutes, regardless of wavelength (nm) ranges tested: blue (~420–495), green (~510–575), and red (~600–700), with green light manifesting the strongest impact. Following shifting of cultures back into the incubator (darkness), cardiac clusters regained beatings within a few hours. The observed light-induced contractility-inhibition effect was intrinsic to cardiomyocytes and not due to interference from other cell types. Also, this was not influenced by any physicochemical parameters or intracellular EGFP expression. Interestingly, the light-induced cardiomyocyte contractility inhibition was accompanied by increased intracellular reactive oxygen species (ROS), which could be abolished in the presence of N-acetylcysteine (ROS quencher). Besides, the increased intracardiomyocyte ROS levels were incidental to the inhibition of calcium transients and suppression of mitochondrial activity, both being essential for sarcomere function. To the best of our knowledge, ours is the first report to demonstrate the monochromatic light-mediated inhibition of contractions of cardiomyocytes with no apparent loss of cell viability and contractility. Our findings have implications in cardiac cell biology context in terms of 1) mechanistic insights into light impact on cardiomyocyte contraction, 2) potential use in laser beam-guided (cardiac) microsurgery, photo-optics-dependent medical diagnostics, 3) transient cessation of hearts during coronary artery bypass grafting, and 4) functional preservation of hearts for transplantation.

scholarly journals Herbal Extract from Codonopsis pilosula (Franch.) Nannf. Enhances Cardiogenic Differentiation and Improves the Function of Infarcted Rat Hearts

Life ◽  
2021 ◽  
Vol 11 (5) ◽  
pp. 422
Author(s):  
Jieh-Neng Wang ◽  
Chung-Dann Kan ◽  
Lain-Tze Lee ◽  
Lynn L. H. Huang ◽  
Ya-Li Hsiao ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Herbal Extract ◽  
Egfp Expression ◽  
Herbal Extracts ◽  
Es Cell ◽  
Codonopsis Pilosula ◽  
Cardiogenic Differentiation

Background: The roots of Codonopsis pilosula (Franch.) Nannf. have been used in traditional Chinese medicine for treating cardiovascular disease. In the current study, we aimed to discover herbal extracts from C. pilosula that are capable of improving cardiac function of infarcted hearts to develop a potential therapeutic approach. Methods: A mouse embryonic stem (ES) cell-based model with an enhanced green fluorescent protein (eGFP) reporter driven by a cardiomyocyte-specific promoter, the α-myosin heavy chain, was constructed to evaluate the cardiogenic activity of herbal extracts. Then, herbal extracts from C. pilosula with cardiogenic activity based on an increase in eGFP expression during ES cell differentiation were further tested in a rat myocardial infarction model with left anterior descending artery (LAD) ligation. Cardiac function assessments were performed using echocardiography, 1, 3, and 6 weeks post LAD ligation. Results: The herbal extract 417W from C. pilosula was capable of enhancing cardiogenic differentiation in mouse ES cells in vitro. Echocardiography results in the LAD-ligated rat model revealed significant improvements in the infarcted hearts at least 6 weeks after 417W treatment that were determined based on left ventricle fractional shortening (FS), fractional area contraction (FAC), and ejection fraction (EF). Conclusions: The herbal extract 417W can enhance the cardiogenic differentiation of ES cells and improve the cardiac function of infarcted hearts.

scholarly journals Transcription Factor Lbx1 Expression in Mouse Embryonic Stem Cell-Derived Phenotypes

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Stefanie Schmitteckert ◽  
Cornelia Ziegler ◽  
Liane Kartes ◽  
Alexandra Rolletschek
Keyword(s):  
Transcription Factor ◽  
Developmental Stages ◽  
Troponin T ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Cardiac Cells ◽  
Cell Stage

Transcription factor Lbx1 is known to play a role in the migration of muscle progenitor cells in limb buds and also in neuronal determination processes. In addition, involvement of Lbx1 in cardiac neural crest-related cardiogenesis was postulated. Here, we used mouse embryonic stem (ES) cells which have the capacity to develop into cells of all three primary germ layers. Duringin vitrodifferentiation, ES cells recapitulate cellular developmental processes and gene expression patterns of early embryogenesis. Transcript analysis revealed a significant upregulation ofLbx1at the progenitor cell stage. Immunofluorescence staining confirmed the expression of Lbx1 in skeletal muscle cell progenitors and GABAergic neurons. To verify the presence of Lbx1 in cardiac cells, triple immunocytochemistry of ES cell-derived cardiomyocytes and a quantification assay were performed at different developmental stages. Colabeling of Lbx1 and cardiac specific markers troponin T, α-actinin, GATA4, and Nkx2.5 suggested a potential role in early myocardial development.

scholarly journals Multipotent fetal-derived Cdx2 cells from placenta regenerate the heart

2019 ◽  
pp. 201811827 ◽  
Author(s):  
Sangeetha Vadakke-Madathil ◽  
Gina LaRocca ◽  
Koen Raedschelders ◽  
Jesse Yoon ◽  
Sarah J. Parker ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Contractile Function ◽  
Es Cells ◽  
Embryonic Stem ◽  
Lineage Tracing ◽  
Cardiac Differentiation ◽  
Cell Based Therapy ◽  
Allogeneic Cell Therapy

The extremely limited regenerative potential of adult mammalian hearts has prompted the need for novel cell-based therapies that can restore contractile function in heart disease. We have previously shown the regenerative potential of mixed fetal cells that were naturally found migrating to the injured maternal heart. Exploiting this intrinsic mechanism led to the current hypothesis that Caudal-type homeobox-2 (Cdx2) cells in placenta may represent a novel cell type for cardiac regeneration. Using a lineage-tracing strategy, we specifically labeled fetal-derived Cdx2 cells with enhanced green fluorescent protein (eGFP). Cdx2-eGFP cells from end-gestation placenta were assayed for cardiac differentiation in vitro and in vivo using a mouse model of myocardial infarction. We observed that these cells differentiated into spontaneously beating cardiomyocytes (CMs) and vascular cells in vitro, indicating multipotentiality. When administered via tail vein to infarcted wild-type male mice, they selectively and robustly homed to the heart and differentiated to CMs and blood vessels, resulting in significant improvement in contractility as noted by MRI. Proteomics and immune transcriptomics studies of Cdx2-eGFP cells compared with embryonic stem (ES) cells reveal that they appear to retain “stem”-related functions of ES cells but exhibit unique signatures supporting roles in homing and survival, with an ability to evade immune surveillance, which is critical for cell-based therapy. Cdx2-eGFP cells may potentially represent a therapeutic advance in allogeneic cell therapy for cardiac repair.

scholarly journals 4D cell biology: big data image analytics and lattice light-sheet imaging reveal dynamics of clathrin-mediated endocytosis in stem cell–derived intestinal organoids

2018 ◽  
Vol 29 (24) ◽  
pp. 2959-2968 ◽  
Author(s):  
Johannes Schöneberg ◽  
Daphné Dambournet ◽  
Tsung-Li Liu ◽  
Ryan Forster ◽  
Dirk Hockemeyer ◽  
...  
Keyword(s):  
Big Data ◽  
Stem Cell ◽  
Adaptive Optics ◽  
Cell Biology ◽  
Fluorescent Protein ◽  
Embryonic Stem ◽  
Light Sheet ◽  
Data Sets ◽  
Intestinal Epithelial ◽  
Imaging Data

New methods in stem cell 3D organoid tissue culture, advanced imaging, and big data image analytics now allow tissue-scale 4D cell biology, but currently available analytical pipelines are inadequate for handing and analyzing the resulting gigabytes and terabytes of high-content imaging data. We expressed fluorescent protein fusions of clathrin and dynamin2 at endogenous levels in genome-edited human embryonic stem cells, which were differentiated into hESC-derived intestinal epithelial organoids. Lattice light-sheet imaging with adaptive optics (AO-LLSM) allowed us to image large volumes of these organoids (70 × 60 × 40 µm xyz) at 5.7 s/frame. We developed an open-source data analysis package termed pyLattice to process the resulting large (∼60 Gb) movie data sets and to track clathrin-mediated endocytosis (CME) events. CME tracks could be recorded from ∼35 cells at a time, resulting in ∼4000 processed tracks per movie. On the basis of their localization in the organoid, we classified CME tracks into apical, lateral, and basal events and found that CME dynamics is similar for all three classes, despite reported differences in membrane tension. pyLattice coupled with AO-LLSM makes possible quantitative high temporal and spatial resolution analysis of subcellular events within tissues.

scholarly journals Specific Double-Stranded RNA Interference in Undifferentiated Mouse Embryonic Stem Cells

2001 ◽  
Vol 21 (22) ◽  
pp. 7807-7816 ◽  
Author(s):  
Shicheng Yang ◽  
Stephen Tutton ◽  
Eric Pierce ◽  
Kyonggeun Yoon
Keyword(s):  
Gene Expression ◽  
Mammalian Cells ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Transient Transfection ◽  
Interferon Response ◽  
Green Fluorescent Protein Gene ◽  
Double Stranded Rna ◽  
Rnai Activity

ABSTRACT Specific mRNA degradation mediated by double-stranded RNA (dsRNA) interference (RNAi) is a powerful way of suppressing gene expression in plants, nematodes, and fungal, insect, and protozoan systems. However, only a few cases of RNAi have been reported in mammalian systems. Here, we investigated the feasibility of the RNAi strategy in several mammalian cells by using the enhanced green fluorescent protein gene as a target, either by in situ production of dsRNA from transient transfection of a plasmid harboring a 547-bp inverted repeat or by direct transfection of dsRNA made by in vitro transcription. Several mammalian cells including differentiated embryonic stem (ES) cells did not exhibit specific RNAi in transient transfection. This long dsRNA, however, was capable of inducing a sequence-specific RNAi for the episomal and chromosomal target gene in undifferentiated ES cells. dsRNA at 8.3 nM decreased the cognate gene expression up to 70%. However, RNAi activity was not permanent because it was more pronounced in early time points and diminished 5 days after transfection. Thus, undifferentiated ES cells may lack the interferon response, similar to mouse embryos and oocytes. Regardless of their apparent RNAi activity, however, cytoplasmic extracts from mammalian cells produced a small RNA of 21 to 22 nucleotides from the long dsRNA. Our results suggest that mammalian cells may possess RNAi activity but nonspecific activation of the interferon response by longer dsRNA may mask the specific RNAi. The findings offer an opportunity to use dsRNA for inhibition of gene expression in ES cells to study differentiation.

183 DEVELOPMENT OF PORCINE EMBRYOS MICROINJECTED WITH PORCINE EMBRYONIC GERM CELLS

2006 ◽  
Vol 18 (2) ◽  
pp. 199
Author(s):  
C.-H. Park ◽  
S.-G. Lee ◽  
D.-H. Choi ◽  
M.-G. Kim ◽  
C. K. Lee
Keyword(s):  
Germ Cells ◽  
Fluorescent Protein ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Green Fluorescent Protein Gene ◽  
Cleavage Stage ◽  
Allocation Pattern

Embryonic germ (EG) cells, derived from primordial germ cells in the developing fetus, are similar to embryonic stem (ES) cells in terms of expression pattern of undifferentiated markers and their ability to colonize both the somatic and the germ cell lines following injection into a host blastocyst, which has been proven in mouse. Several studies using porcine EG cells have shown that it is possible to produce somatic chimeras after blastocyst injection. However, not only was the degree of reported chimerism low, but also there has been no report about the fate of injected EG cells in porcine blastocysts. This study was designed to observe the distribution pattern of porcine EG cells in chimeric blastocyst after injection into cleavage-stage porcine embryos. To ascertain development of microinjected porcine embryos with EG cells, 10 to 15 EG cells were injected into cleavage stage of in vitro fertilized embryos and cultured up to blastocyst. Also, porcine EG cells were labeled with DiO (Invitrogen, Carlsbad, CA) on the cell membrane or transfected with green fluorescent protein gene to observe whether the EG cells injected in the host embryo would incorporate into the inner cell mass (ICM) or trophectoderm (TE). Chimeric embryos were produced and allowed to develop into blastocysts to investigate the injected EG cells would come to lie in ICM and/or TE of the blastocyst, by scoring their position. In result, developmental rate was similar in all treatments. In all treatments, EG cells were mainly allocated in both ICM and TE of the chimeric blastocysts. These results suggest that examining the allocation pattern of injected EG cells, maintained pluripotency in vitro, could provide clues of differentiation process in vivo. Furthermore, to enhance the allocation of EG cells into the embryonic lineage, it would be required to optimize the culture condition for EG cells as well as embryos. Further experiment are needed to determine whether the injected EG cells could maintain their properties throughout the environment in the embryonic development in vitro. Table 1. Distribution of the porcine EG cells microinjected into cleavage-stage embryos

Specificity of Smad Signaling during Primitive Erythropoiesis.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2785-2785
Author(s):  
Brian T. Zafonte ◽  
Tara L. Huber ◽  
Gordon Keller ◽  
Todd Evans
Keyword(s):  
Embryoid Body ◽  
Biological Activities ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Dominant Negative ◽  
Hematopoietic Progenitors ◽  
Transcript Levels ◽  
Wide Range ◽  
Primitive Erythropoiesis

Abstract Bone morphogenetic proteins (BMPs) comprise a sub-family of TGF-beta-like molecules that exert a wide range of biological activities during development, and are essential for normal hematopoiesis. However, the precise stage in development that BMP signaling regulates hematopoiesis is not defined. Three proteins, Smad1, Smad5, and Smad8 transmit BMP signals to the nucleus to activate the expression of hematopoietic-specific transcription factors. These Smads are homologous in their sequences, and appear to be regulated similarly, however their specificity in regulating hematopoiesis remains undefined. Although Smad proteins are regulated post-translationally, their expression is also under transcriptional control during development. We examined the specificity of Smad1/5/8 activity in the context of primitive erythropoiesis, using the mouse embryonic stem cell /embryoid body (ES/EB) system. We exploited ES cells with GFP targeted to the brachyury locus, in order to identify specific sub-sets of progenitors. Smad1 transcript levels are initially upregulated as ES cells become fated to mesoderm and hematopoietic progenitors, but the levels are significantly decreased in cells derived from differentiating primitive erythroid colonies. In contrast, Smad5 transcript levels show the opposite profile, being more correlated with erythroid differentiation. To directly assess the role of these Smads during erythropoiesis, their activity is being manipulated in ES cells during the commitment phases of embryonic hematopoiesis. For this purpose, inducible ES cell lines were generated capable of forcing the expression of wildtype Smad1 or Smad5, or a dominant-negative isoform of Smad5, at any stage of ES/EB development. Colony assays were used to analyze quantitatively the hematopoietic potential of these cells. Forced expression of Smad1 results in a marked increase in primitive red blood cell colony formation as compared to control ES cells. Maintenance of Smad1 expression does not appear to inhibit terminal differentiation. Based on a time-study of the induction, the effect on erythoid colonies could be due to expansion of earlier progenitors. Current experiments using the in vitro blast assay are examining the direct effect of Smad1 expression on earlier (hemangioblast) development. This data, and analogous analyses of cells induced to express Smad5 or the dominant-negative Smad isoform are in progress and will be presented. These studies should facilitate our understanding of the specificity of BMP-regulated Smads during commitment and differentiation of embryonic stem cells and hematopoietic progenitors.

scholarly journals Nanog fluctuations in ES cells highlight the problem of measurement in cell biology

2016 ◽  
Author(s):  
Rosanna C G Smith ◽  
Patrick S Stumpf ◽  
Sonya J Ridden ◽  
Aaron Sim ◽  
Sarah Filippi ◽  
...  
Keyword(s):  
Cell Lines ◽  
Cell Biology ◽  
Measurement Problem ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Genetically Engineered ◽  
Live Cells ◽  
Reporter Cell ◽  
Nanog Expression

A number of important pluripotency regulators, including the transcription factor Nanog, are observed to fluctuate stochastically in individual embryonic stem (ES) cells. By transiently priming cells for commitment to different lineages, these fluctuations are thought to be important to the maintenance of, and exit from, pluripotency. However, since temporal changes in intracellular protein abundances cannot be measured directly in live cells, these fluctuations are typically assessed using genetically engineered reporter cell lines that produce a fluorescent signal as a proxy for protein expression. Here, using a combination of mathematical modeling and experiment, we show that there are unforeseen ways in which widely used reporter strategies can systemically disturb the dynamics they are intended to monitor, sometimes giving profoundly misleading results. In the case of Nanog we show how genetic reporters can compromise the behavior of important pluripotency-sustaining positive feedback loops, and induce a bifurcation in the underlying dynamics that gives rise to heterogeneous Nanog expression patterns in reporter cell lines that are not representative of the wild-type. These findings help explain the range of published observations of Nanog variability and highlight a fundamental measurement problem in cell biology.

scholarly journals A Rapid and Highly Efficient Genotyping Pipeline for Screening Gene Targeted Mouse Embryonic Stem Cells

2021 ◽  
Author(s):  
Roger Caothien ◽  
Charles Yu ◽  
Lucinda Tam ◽  
Robert Newman ◽  
Brian Nakao ◽  
...  
Keyword(s):  
Animal Models ◽  
Gene Targeting ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Expression Patterns ◽  
Embryonic Stem ◽  
Rapid Screening ◽  
Es Cell ◽  
Screening Process ◽  
Targeting Vector

Abstract Gene targeting in mouse ES cells replaces or modifies genes of interest; conditional alleles, reporter knock-ins, and amino acid changes are common examples of how gene targeting is used. For example, enhanced green fluorescent protein or Cre recombinase is placed under the control of endogenous genes to define promoter expression patterns. The most important step in the process is to demonstrate that a gene targeting vector is correctly integrated in the genome at the desired chromosomal location. The rapid identification of correctly targeted ES cell clones is facilitated by proper targeting vector construction, rapid screening procedures, and advances in cell culture. The addition of magnetic activated cell sorting (MACS) technology and multiplex droplet digital PCR (ddPCR) to the ES cell screening process can achieve a greater than 60% assurance that ES clones are correctly targeted. In a further refinement of the process, drug selection cassettes are removed from ES cells with adenovirus technology. This improved workflow reduces the time needed to generate preclinical animal models. Faster access to animal models for therapeutic target identification and experimental validation can accelerate the development of therapies for human disease.

High-content screening of small compounds on human embryonic stem cells

2010 ◽  
Vol 38 (4) ◽  
pp. 1046-1050 ◽  
Author(s):  
Ivana Barbaric ◽  
Paul J. Gokhale ◽  
Peter W. Andrews
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
High Throughput ◽  
Cell Biology ◽  
Es Cells ◽  
Embryonic Stem ◽  
High Content Screening ◽  
Cellular Models ◽  
Compound Libraries

Human ES (embryonic stem) cells and iPS (induced pluripotent stem) cells have been heralded as a source of differentiated cells that could be used in the treatment of degenerative diseases, such as Parkinson's disease or diabetes. Despite the great potential for their use in regenerative therapy, the challenge remains to understand the basic biology of these remarkable cells, in order to differentiate them into any functional cell type. Given the scale of the task, high-throughput screening of agents and culture conditions offers one way to accelerate these studies. The screening of small-compound libraries is particularly amenable to such high-throughput methods. Coupled with high-content screening technology that enables simultaneous assessment of multiple cellular features in an automated and quantitative way, this approach is proving powerful in identifying both small molecules as tools for manipulating stem cell fates and novel mechanisms of differentiation not previously associated with stem cell biology. Such screens performed on human ES cells also demonstrate the usefulness of human ES/iPS cells as cellular models for pharmacological testing of drug efficacy and toxicity, possibly a more imminent use of these cells than in regenerative medicine.

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