scholarly journals Microfluidic platform for live cell imaging of 3D cultures with clone retrieval

2020 ◽  
Author(s):  
Carla Mulas ◽  
Andrew C Hodgson ◽  
Timo N Kohler ◽  
Chibeza C Agley ◽  
Florian Hollfelder ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Encapsulation ◽  
Live Imaging ◽  
Live Cells ◽  
Small Scale ◽  
Reporter Expression ◽  
Microfluidic Platform ◽  
Functional Assays ◽  
Hydrogel Matrix

ABSTRACTCombining live imaging with the ability to retrieve individual cells of interest remains a technical challenge. These combined methods are of particular interest when studying highly dynamic or transient, asynchronous or heterogeneous cell biological and developmental processes. Here we present a method to encapsulate live cells in a 3D hydrogel matrix, via droplet compartmentalisation. Using a small-scale screen, we optimised matrix conditions for the culture and multilineage differentiation of mouse embryonic stem (ES) cells. Moreover, we designed a custom microfluidic platform that is compatible with live imaging. With this platform we are able to retain or extract individual bead/droplets by media flow only, obviating the need for enzymatic cell removal from the platform. We show that we can differentiate mES cells, monitor reporter expression by live imaging, and retrieve individual droplets for functional assays, correlating reporter expression with functional response. Overall, we present a highly flexible 3D cell encapsulation and microfluidic platform that enables both monitoring of cellular dynamics and retrieval for molecular and functional assays.

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 Production of interspecies somatic/pluripotent heterokaryons using polyethylene glycol (PEG) and selection by imaging flow cytometry for the study of nuclear reprogramming

2019 ◽  
Author(s):  
M. Cristina Villafranca ◽  
Melissa R. Makris ◽  
Maria Jesus Garrido Bauerle ◽  
Roderick V. Jensen ◽  
Willard H. Eyestone
Keyword(s):  
Flow Cytometry ◽  
Polyethylene Glycol ◽  
Mammalian Cells ◽  
Cell Damage ◽  
Es Cells ◽  
Embryonic Stem ◽  
Live Cells ◽  
Nuclear Reprogramming ◽  
Somatic Nucleus ◽  
Nih 3T3

ABSTRACTFusion of somatic cells to pluripotent cells such as mouse embryonic stem (ES) cells induces reprogramming of the somatic nucleus, and can be used to study the effect of trans-acting factors from the pluripotent cell on the somatic nucleus. Moreover, fusion of cells from different species permits the identification of the transcriptome of each cell, so the gene expression changes can be monitored. However, fusion only happens in a small proportion of the cells exposed to fusogenic conditions, hence the need for a protocol that produces high fusion rate with minimal cell damage, coupled with a method capable of identifying and selecting fusion events from the bulk of the cells. Polyethylene glycol (PEG) is a polymer of repeated ethylene oxide units known to induce cell fusion within a certain range of molecular weight. Here, we describe a method to induce formation of bi-species heterokaryons from adherent mammalian cells, which can then be specifically labeled and selected using live cell immunostaining and a combination of imaging and traditional flow cytometry. First, we tested several PEG-based fusion conditions to optimize a protocol to consistently produce both mouse NIH/3T3 fibroblast and primary bovine fetal fibroblast (bFF) homokaryons. Initially, we obtained 7.28% of NIH/3T3 homokaryons when using 50% PEG 1500. Addition of 10% of DMSO to the PEG solution increased the percentage of NIH/3T3 homokaryons to 11.71%. In bFFs, treatment with 50% PEG 1500 plus 10% DMSO produced 11.05% of homokaryons. We then produced interspecies heterokaryons by fusing mouse embryonic stem (mES) cells to bFFs. To identify bi-species fusion products, heterokaryons were labeled using indirect immunostaining in live cells and selected using imaging (Amnis ImageStream Mark II) and traditional (BD FACSAria I) flow cytometry. Heterokaryons selected with this method produced ES cell-like colonies when placed back in culture. The method described here can also be combined with downstream applications such as nucleic acid isolation for RT-PCR and RNA-seq, and used as a tool to study cellular processes in which the effect of trans-acting factors is relevant, such as in nuclear reprogramming.

scholarly journals A Haplolethal Locus Uncovered by Deletions in the Mouse t Complex

Genetics ◽  
2002 ◽  
Vol 160 (2) ◽  
pp. 675-682
Author(s):  
Victoria L Browning ◽  
Rebecca A Bergstrom ◽  
Sandra Daigle ◽  
John C Schimenti
Keyword(s):  
Gene Dosage ◽  
Es Cells ◽  
Embryonic Stem ◽  
Chromosome 17 ◽  
Mammalian Development ◽  
T Complex ◽  
Segmental Aneuploidy ◽  
Systematic Exploration ◽  
Radiation Induced ◽  
Altered Gene

Abstract Proper levels of gene expression are important for normal mammalian development. Typically, altered gene dosage caused by karyotypic abnormalities results in embryonic lethality or birth defects. Segmental aneuploidy can be compatible with life but often results in contiguous gene syndromes. The ability to manipulate the mouse genome allows the systematic exploration of regions that are affected by alterations in gene dosage. To explore the effects of segmental haploidy in the mouse t complex on chromosome 17, radiation-induced deletion complexes centered at the Sod2 and D17Leh94 loci were generated in embryonic stem (ES) cells. A small interval was identified that, when hemizygous, caused specific embryonic lethal phenotypes (exencephaly and edema) in most fetuses. The penetrance of these phenotypes was background dependent. Additionally, evidence for parent-of-origin effects was observed. This genetic approach should be useful for identifying genes that are imprinted or whose dosage is critical for normal embryonic development.

scholarly journals The chromosomal protein SMCHD1 regulates DNA methylation and the 2c-like state of embryonic stem cells by antagonizing TET proteins

2021 ◽  
Vol 7 (4) ◽  
pp. eabb9149
Author(s):  
Zhijun Huang ◽  
Jiyoung Yu ◽  
Wei Cui ◽  
Benjamin K. Johnson ◽  
Kyunggon Kim ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Homeobox Gene ◽  
Dna Demethylation ◽  
Chromosomal Protein ◽  
Dna Hypomethylation ◽  
Tet Proteins ◽  
Target Sites ◽  
Structural Maintenance Of Chromosomes ◽  
Hinge Domain

5-Methylcytosine (5mC) oxidases, the ten-eleven translocation (TET) proteins, initiate DNA demethylation, but it is unclear how 5mC oxidation is regulated. We show that the protein SMCHD1 (structural maintenance of chromosomes flexible hinge domain containing 1) is found in complexes with TET proteins and negatively regulates TET activities. Removal of SMCHD1 from mouse embryonic stem (ES) cells induces DNA hypomethylation, preferentially at SMCHD1 target sites and accumulation of 5-hydroxymethylcytosine (5hmC), along with promoter demethylation and activation of the Dux double-homeobox gene. In the absence of SMCHD1, ES cells acquire a two-cell (2c) embryo–like state characterized by activation of an early embryonic transcriptome that is substantially imposed by Dux. Using Smchd1/Tet1/Tet2/Tet3 quadruple-knockout cells, we show that DNA demethylation, activation of Dux, and other genes upon SMCHD1 loss depend on TET proteins. These data identify SMCHD1 as an antagonist of the 2c-like state of ES cells and of TET-mediated DNA demethylation.

scholarly journals Retinoic acid signaling is critical during the totipotency window in early mammalian development

2021 ◽  
Author(s):  
Ane Iturbide ◽  
Mayra L. Ruiz Tejeda Segura ◽  
Camille Noll ◽  
Kenji Schorpp ◽  
Ina Rothenaigner ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Regulatory Networks ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mammalian Development ◽  
Cell Stage ◽  
Cell Potential ◽  
Cellular Models ◽  
Early Embryos ◽  
Genome Activation

AbstractTotipotent cells hold enormous potential for regenerative medicine. Thus, the development of cellular models recapitulating totipotent-like features is of paramount importance. Cells resembling the totipotent cells of early embryos arise spontaneously in mouse embryonic stem (ES) cell cultures. Such ‘2-cell-like-cells’ (2CLCs) recapitulate 2-cell-stage features and display expanded cell potential. Here, we used 2CLCs to perform a small-molecule screen to identify new pathways regulating the 2-cell-stage program. We identified retinoids as robust inducers of 2CLCs and the retinoic acid (RA)-signaling pathway as a key component of the regulatory circuitry of totipotent cells in embryos. Using single-cell RNA-seq, we reveal the transcriptional dynamics of 2CLC reprogramming and show that ES cells undergo distinct cellular trajectories in response to RA. Importantly, endogenous RA activity in early embryos is essential for zygotic genome activation and developmental progression. Overall, our data shed light on the gene regulatory networks controlling cellular plasticity and the totipotency program.

scholarly journals The dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

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 Epigenetic Regulation of Mesenchymal Stem Cells: A Focus on Osteogenic and Adipogenic Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-18 ◽  
Author(s):  
Chad M. Teven ◽  
Xing Liu ◽  
Ning Hu ◽  
Ni Tang ◽  
Stephanie H. Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Epigenetic Regulation ◽  
Adult Stem Cells ◽  
Es Cells ◽  
Adipogenic Differentiation ◽  
Embryonic Stem ◽  
Cell Types ◽  
Differentiation Potential ◽  
Msc Differentiation

Stem cells are characterized by their capability to self-renew and terminally differentiate into multiple cell types. Somatic or adult stem cells have a finite self-renewal capacity and are lineage-restricted. The use of adult stem cells for therapeutic purposes has been a topic of recent interest given the ethical considerations associated with embryonic stem (ES) cells. Mesenchymal stem cells (MSCs) are adult stem cells that can differentiate into osteogenic, adipogenic, chondrogenic, or myogenic lineages. Owing to their ease of isolation and unique characteristics, MSCs have been widely regarded as potential candidates for tissue engineering and repair. While various signaling molecules important to MSC differentiation have been identified, our complete understanding of this process is lacking. Recent investigations focused on the role of epigenetic regulation in lineage-specific differentiation of MSCs have shown that unique patterns of DNA methylation and histone modifications play an important role in the induction of MSC differentiation toward specific lineages. Nevertheless, MSC epigenetic profiles reflect a more restricted differentiation potential as compared to ES cells. Here we review the effect of epigenetic modifications on MSC multipotency and differentiation, with a focus on osteogenic and adipogenic differentiation. We also highlight clinical applications of MSC epigenetics and nuclear reprogramming.

scholarly journals Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽  
2011 ◽  
Vol 152 (7) ◽  
pp. 2870-2882 ◽  
Author(s):  
Unmesh Jadhav ◽  
J. Larry Jameson
Keyword(s):  
Target Genes ◽  
De Novo ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Culture Conditions ◽  
Steroidogenic Factor 1 ◽  
Lineage Differentiation ◽  
And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

Sign in / Sign up

Export Citation Format

Share Document