ROCK inhibitor Y-27632 enhances the survivability of dissociated buffalo (Bubalus bubalis) embryonic stem cell-like cells

2013 ◽  
Vol 25 (2) ◽  
pp. 446 ◽  
Author(s):  
Ruchi Sharma ◽  
Aman George ◽  
Manmohan S. Chauhan ◽  
Suresh Singla ◽  
Radhey S. Manik ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Es Cells ◽  
Single Cells ◽  
Formation Rate ◽  
Embryonic Stem ◽  
Rho Kinase ◽  
Specific Inhibitor ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Rock Inhibitor

This study investigated the effects of supplementation of culture medium with 10 μM Y-27632, a specific inhibitor of Rho kinase activity, for 6 days on self-renewal of buffalo embryonic stem (ES) cell-like cells at Passage 50–80. Y-27632 increased mean colony area (P < 0.05) although it did not improve their survival. It decreased OCT4 expression (P < 0.05), increased NANOG expression (P < 0.05), but had no effect on SOX2 expression. It also increased expression of anti-apoptotic gene BCL-2 (P < 0.05) and decreased that of pro-apoptotic genes BAX and BID (P < 0.05). It increased plating efficiency of single-cell suspensions of ES cells (P < 0.05). Following vitrification, the presence of Y-27632 in the vitrification solution or thawing medium or both did not improve ES cell colony survival. However, following seeding of clumps of ES cells transfected with pAcGFP1N1 carrying green fluorescent protein (GFP), Y-27632 increased colony formation rate (P < 0.01). ES cell colonies that formed in all Y-27632-supplemented groups were confirmed for expression of pluripotency markers alkaline phosphatase, SSEA-4 and TRA-1–60, and for their ability to generate embryoid bodies containing cells that expressed markers of ectoderm, mesoderm and endoderm. In conclusion, Y-27632 improves survival of buffalo ES cells under unfavourable conditions such as enzymatic dissociation to single cells or antibiotic-assisted selection after transfection, without compromising their pluripotency.

scholarly journals Monkey Embryonic Stem Cell Lines Expressing Green Fluorescent Protein

2002 ◽  
Vol 11 (7) ◽  
pp. 631-635 ◽  
Author(s):  
Tatsuyuki Takada ◽  
Yutaka Suzuki ◽  
Yasushi Kondo ◽  
Nae Kadota ◽  
Kinji Kobayashi ◽  
...  
Keyword(s):  
Green Fluorescent Protein ◽  
Cell Transplantation ◽  
Cell Lines ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Green Fluorescent

The major limitation of nonhuman primate (NHP) embryonic stem (ES) cell research is inefficient genetic modification and limited knowledge of differentiation mechanisms. A genetically modified NHP-ES cell with biomarkers, such as green fluorescent protein (GFP), that allow noninvasive monitoring of transgenic cells, is a useful tool to study cell differentiation control during preimplantation and fetal development, which also plays a crucial role in the development of cell transplantation medicine. Here we report the establishment of transgenic NHP-ES cell lines that express GFP without jeopardizing their pluripotency, which was confirmed by in vitro and in vivo differentiation. These GFP-expressing ES cells reproducibly differentiated into embryoid bodies, neural cells, and cardiac myocytes. They formed teratoma composed of tissues derived from the three embryonic germ layers when transplanted into severe combined immunodeficient disease (SCID) mice. GFP expression was maintained in these differentiated cells, suggesting that these cells were useful for cell transplantation experiments. Furthermore, we showed that these ES cells have the ability to form chimeric blastocysts by introducing into the early preimplantation stage NHP embryo.

Transplantation of embryonic stem cells improves cardiac function in postinfarcted rats

2002 ◽  
Vol 92 (1) ◽  
pp. 288-296 ◽  
Author(s):  
Jiang-Yong Min ◽  
Yinke Yang ◽  
Kimber L. Converso ◽  
Lixin Liu ◽  
Qin Huang ◽  
...  
Keyword(s):  
Cardiac Function ◽  
Cell Transplantation ◽  
Troponin I ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Control Group ◽  
Positive Staining ◽  
Es Cell ◽  
Intramyocardial Injection

Massive loss of cardiac myocytes after myocardial infarction (MI) is a common cause of heart failure. The present study was designed to investigate the improvement of cardiac function in MI rats after embryonic stem (ES) cell transplantation. MI in rats was induced by ligation of the left anterior descending coronary artery. Cultured ES cells used for cell transplantation were transfected with the marker green fluorescent protein (GFP). Animals in the treated group received intramyocardial injection of ES cells in injured myocardium. Compared with the MI control group injected with an equivalent volume of the cell-free medium, cardiac function in ES cell-implanted MI animals was significantly improved 6 wk after cell transplantation. The characteristic phenotype of engrafted ES cells was identified in implanted myocardium by strong positive staining to sarcomeric α-actin, cardiac α-myosin heavy chain, and troponin I. GFP-positive cells in myocardium sectioned from MI hearts confirmed the survival and differentiation of engrafted cells. In addition, single cells isolated from cell-transplanted MI hearts showed rod-shaped GFP-positive myocytes with typical striations. The present data demonstrate that ES cell transplantation is a feasible and novel approach to improve ventricular function in infarcted failing hearts.

X chromosome retains the memory of its parental origin in murine embryonic stem cells

Development ◽  
1993 ◽  
Vol 119 (3) ◽  
pp. 813-821 ◽  
Author(s):  
T. Tada ◽  
M. Tada ◽  
N. Takagi
Keyword(s):  
X Chromosome ◽  
Polar Region ◽  
Biochemical Study ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Parental Origin ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Preferential Inactivation

A cytogenetic and biochemical study of balloon-like cystic embryoid bodies, formed by newly established embryonic stem (ES) cell lines having a cytogenetically or genetically marked X chromosome, revealed that the paternally derived X chromosome was inactivated in the majority of cells in the yolk sac-like mural region consisting of the visceral endoderm and mesoderm. The nonrandomness was less evident in the more solid polar region containing the ectodermal vesicle, mesoderm and visceral endoderm. Since the same was true in embryoid bodies derived from ES cells at the 30th subculture generation, it was concluded that the imprinting responsible for the preferential inactivation of the paternal X chromosome that was limited to non-epiblast cells of the female mouse embryos, was stably maintained in undifferentiated ES cells. Differentiating epiblast cells should be able to erase or avoid responding to the imprint.

Filamin A Controls Embryonic Stem Cell Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4599-4599
Author(s):  
Taisuke Kanaji ◽  
Takashi Okamura ◽  
Peter J. Newman
Keyword(s):  
Cell Differentiation ◽  
Signaling Pathways ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Actin Binding ◽  
Embryonic Stem Cell Differentiation ◽  
Filamin A ◽  
Es Cell

Abstract Abstract 4599 Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells. Disclosures: Newman: Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Lentivirus Vector Gene Expression during ES Cell-Derived Hematopoietic Development In Vitro

2000 ◽  
Vol 74 (22) ◽  
pp. 10778-10784 ◽  
Author(s):  
Isao Hamaguchi ◽  
Niels-Bjarne Woods ◽  
Ioannis Panagopoulos ◽  
Elisabet Andersson ◽  
Hanna Mikkola ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Experimental System ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Lentivirus Vector ◽  
Gfp Gene ◽  
Development In Vitro

ABSTRACT The murine embryonal stem (ES) cell virus (MESV) can express transgenes from the long terminal repeat (LTR) promoter/enhancer in undifferentiated ES cells, but expression is turned off upon differentiation to embryoid bodies (EBs) and hematopoietic cells in vitro. We examined whether a human immunodeficiency virus type 1-based lentivirus vector pseudotyped with the vesicular stomatitis virus G protein (VSV-G) could transduce ES cells efficiently and express the green fluorescent protein (GFP) transgene from an internal phosphoglycerate kinase (PGK) promoter throughout development to hematopoietic cells in vitro. An oncoretrovirus vector containing the MESV LTR and the GFP gene was used for comparison. Fluorescence-activated cell sorting analysis of transduced CCE ES cells showed 99.8 and 86.7% GPF-expressing ES cells in the VSV-G-pseudotyped lentivirus (multiplicity of infection [MOI] = 59)- and oncoretrovirus (MOI = 590)-transduced cells, respectively. Therefore, VSV-G pseudotyping of lentiviral and oncoretrovirus vectors leads to efficient transduction of ES cells. Lentivirus vector integration was verified in the ES cell colonies by Southern blot analysis. When the transduced ES cells were differentiated in vitro, expression from the oncoretrovirus LTR was severely reduced or extinct in day 6 EBs and ES cell-derived hematopoietic colonies. In contrast, many lentivirus-transduced colonies, expressing the GFP gene in the undifferentiated state, continued to express the transgene throughout in vitro development to EBs at day 6, and many continued to express in cells derived from hematopoietic colonies. This experimental system can be used to analyze lentivirus vector design for optimal expression in hematopoietic cells and for gain-of-function experiments during ES cell development in vitro.

scholarly journals Tracking the embryonic stem cell transition from ground state pluripotency

2016 ◽  
Author(s):  
Tüzer Kalkan ◽  
Nelly Olova ◽  
Mila Roode ◽  
Carla Mulas ◽  
Heather J. Lee ◽  
...  
Keyword(s):  
Ground State ◽  
Es Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
List Type ◽  
Es Cell ◽  
Genome Wide ◽  
Developmental Progression ◽  
A Genome ◽  
Lineage Priming

SummaryMouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition of ES cells. The population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state exhibit global transcriptome features consistent with features of early post-implantation epiblast and distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a discrete formative phase of pluripotency preparatory to lineage priming.HighlightsThe Rex1 destabilized GFP reporter demarcates naive pluripotency.Exit from the naive state is asynchronous in the population.Transition is relatively acute in individual cells and precedes lineage priming.Transcriptome and DNA methylome reflect events in the pre-gastrulation embryo.

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.

DNA synthesis and multinucleation in embryonic stem cell-derived cardiomyocytes

1995 ◽  
Vol 269 (6) ◽  
pp. H1913-H1921 ◽  
Author(s):  
M. G. Klug ◽  
M. H. Soonpaa ◽  
L. J. Field
Keyword(s):  
Dna Synthesis ◽  
Thymidine Incorporation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Es Cell ◽  
Isolated Cells ◽  
In Vitro System ◽  
Multinucleated Cells

The proliferative capacity of embryonic stem (ES) cell-derived cardiomyocytes was assessed. Enriched preparations of cardiomyocytes were isolated by microdissection of the cardiogenic regions of cultured embryoid bodies. The identity of the isolated cells was established by immunocytology, and mitotic activity was monitored by [3H]thymidine incorporation and pulse-chase experiments. ES-derived cardiomyocytes were mitotically active and predominantly mononucleated at 11 days after cardiogenic induction. By 21 days postinduction, cardiomyocyte DNA synthesis was markedly decreased, with a concomitant increase in the percentage of multinucleated cells. Interestingly, the duration of active cardiomyocyte reduplication in the ES system appeared to be roughly similar to that observed during normal murine cardiogenesis. Given these observations, as well as the genetic tractability of ES cells, ES-derived cardiogenesis might provide a useful in vitro system with which to assess the molecular regulation of the cardiomyocyte cell cycle.

Derivation, Characterization, and In Vitro Differentiation of Canine Embryonic Stem Cells.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4059-4059
Author(s):  
Aravind Ramakrishnan ◽  
Brian Hayes ◽  
Sara R. Fagerlie ◽  
Szczepan Baran ◽  
Michael Harkey ◽  
...  
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Clara Cell ◽  
In Vivo Model ◽  
Large Animal ◽  
Es Cell

Abstract Embryonic stem (ES) cells have created considerable excitement in the last few years due to their unlimited potential to produce cells for tissue repair and replacement. However, a large animal pre-clinical model is necessary to establish the safety and efficacy of ES cell-derived tissue replacement therapy. The canine model has long been used in medical research, has been well established to study adult stem cell transplantation and has been highly predictive of clinical outcomes in humans, more so than rodent models. Given the documented record for extrapolating from dog to man, we hypothesize that the dog would serve as an ideal pre-clinical in vivo model for studying the clinical applications of ESC derived tissue. Eleven putative ES cell lines were initiated from canine blastocysts harvested from natural matings. One line described here, FHDO-7, has been maintained through 34 passages and has many characteristics of ES cells from other species. FHDO-7 cells are alkaline phosphatase positive and express both message and protein for the Oct4 transcription factor. They also express message for Nanog and do not express message for Cdx2 which is associated with trophectoderm. Furthermore, they express a cluster of pluripotency-associated microRNAs (miR-302b, miR-302c and miR-367) that have been found to be characteristic of human and mouse ES cells. The FHDO-7 cells grow on feeder layers of modified mouse embryonic fibroblasts (MEF) as flat colonies that resemble ES cells from mink, a close phylogenetic relative of dog. When cultured in nonadherent plates without feeders the cells form embryoid bodies (EB). Under various culture conditions the EBs give rise to ectoderm-derived neuronal cells expressing β3-tubulin, mesoderm-derived osteocytes producing bone, and endoderm-derived cells expressing alpha feto protein or Clara cell specific protein. These results indicate that FHDO-7 is a pluripotent embryonic stem cell line.

Embryoid Body Defect in Mouse Rps19-Haploinsufficient Embryonic Stem Cell Model of Diamond Blackfan Anemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3093-3093
Author(s):  
Sharon Singh ◽  
Sehba Dsilva ◽  
Jeffrey Michael Lipton ◽  
Steven Ellis ◽  
Johnson M. Liu
Keyword(s):  
Cell Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Gene Trap ◽  
Embryoid Bodies ◽  
Targeted Mutagenesis ◽  
Es Cell ◽  
Chimeric Mice ◽  
Diamond Blackfan Anemia ◽  
Significant Difference

Abstract Diamond Blackfan anemia (DBA) is an inherited bone marrow failure syndrome that is characterized by erythroid hypoplasia, risk of other cytopenias, congenital anomalies and a cancer predisposition. Thus far, all the genes identified as mutated in DBA encode ribosomal proteins (RPS19, RPS17, RPS24, RPL5, RPL11, and RPL35a). In the 25% of DBA patients with RPS19 mutations, haploinsufficiency of RPS19 has been linked to faulty ribosome biogenesis, which ultimately predisposes erythroid precursors to apoptosis through as yet unknown mechanisms. Previous attempts by others to apply targeted mutagenesis to Rps19 were unsuccessful because of compensatory Rps19 expression from the non-targeted allele. We have concentrated our efforts on characterizing the murine Rps19-mutated embryonic stem (ES) cell, S17-10H1, which was generated using a genetrap strategy. The gene-trap vector contains a strong splice acceptor-β-geo cassette-poly A termination, and following insertion, it should cause splicing with the exon upstream and termination at the poly A signal, effectively cutting Rps19 in half. S17-10H1 was sequenced using 3′ RACE (rapid amplification of cDNA ends) to confirm insertion of the vector between exons 2 and 3 of Rps19. PCR with primers against the β-geo sequence was also used to confirm insertion of the gene trap vector into the mutant ES cells. Western blot analysis of two different ES cell samples confirmed at least 50% less Rps19 protein than found in the wild-type parental ES cell line, AK7. The ES cells were subsequently induced to undergo primary differentiation into embryoid bodies (EBs). Although there was no significant difference in the EB size or shape at day 5 of culture, the number of EBs that formed in the mutant cultures was decreased by at least three-fold. Preliminary experiments indicated no obvious morphological differences in day 13 EBs derived from parental or mutant ES cells. We attempted to create chimeric mice by microinjection of the S17-10H1 cell line into 36 blastocysts. Six chimeric mice were set up in mating pairs with C57BL/6J partners. Analysis of more than 60 pups from the 60% chimeric male revealed a lack of germline transmission, possibly indicating that this mutation leads to embryonic lethality or inability to complete gametogenesis. We conclude that this ES cell differentiation model mimics the human disease in leading to Rps19 haploinsufficiency and provides a new and potentially powerful tool that can be used to elucidate molecular mechanisms and test potential therapies in DBA.

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