Caveolin-1 plays important role in EGF-induced migration and proliferation of mouse embryonic stem cells: involvement of PI3K/Akt and ERK

2009 ◽  
Vol 297 (4) ◽  
pp. C935-C944 ◽  
Author(s):  
Jae Hong Park ◽  
Ho Jae Han
Keyword(s):  
Cell Cycle ◽  
Cell Migration ◽  
Kinase Inhibitor ◽  
Thymidine Incorporation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Akt Inhibitor ◽  
Expression Levels ◽  
Caveolin 1

The involvement of caveolin-1 in the regulation of embryonic stem (ES) cell growth by epidermal growth factor (EGF) is by no means clear cut. Thus we examined the relationship between EGF and caveolin-1 in mouse ES cell migration and proliferation. The results revealed that EGF increased Src, caveolin-1, focal adhesion kinase (FAK), Akt, and extracellular signal-regulated kinase-1/2 (ERK) phosphorylation levels. Especially, phosphorylation of caveolin-1 is attenuated by AG1478, herbimycin A (tyrosine kinase inhibitors), and pyrazolopyrimidine 2 (PP2, Src inhibitor) and EGF-induced ERK activation was blocked by PP2, methyl-β-cyclodextrin (MβCD), caveolin-1 small interfering RNA (siRNA), LY-294002 [phosphoinositol-3 kinase inhibitor (PI3K)], and Akt inhibitor. In addition, EGF promoted the cell migration, which was attenuated by PP2, caveolin-1 siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. EGF also increased matrix metalloproteinase (MMP-2) expression levels and EGF-induced MMP2 expression was inhibited by caveolin-1 siRNA, FAK siRNA, LY-294002, Akt inhibitor, and PD-98059. Furthermore, EGF-induced increase of cell cycle proteins expression level and [3H]thymidine incorporation was blocked by MMP inhibitor. EGF also significantly increases [3H]thymidine incorporation and cell number, which were significantly blocked by AG 1478, PP2, MβCD, caveolin-1 siRNA, FAK siRNA, LY-294002, and PD-98059 (ERK inhibitor). EGF-induced increase of protooncogenes (c- fos, c- myc, and c- Jun) and cell cycle regulatory proteins (cyclin D1, CDK4, cyclin E, and CDK2) expression levels were also attenuated by caveolin-1 siRNA and FAK siRNA. In conclusion, these results demonstrated that EGF-induced DNA synthesis and cell migration are mediated by caveolin-1, which is activated by Src, FAK, PI3K/Akt, ERK, and MMP-2 signals in mouse ES cells.

scholarly journals T-type Ca2+ channels in mouse embryonic stem cells: modulation during cell cycle and contribution to self-renewal

2012 ◽  
Vol 302 (3) ◽  
pp. C494-C504 ◽  
Author(s):  
José A. Rodríguez-Gómez ◽  
Konstantín L. Levitsky ◽  
José López-Barneo
Keyword(s):  
Cell Cycle ◽  
Alkaline Phosphatase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Mrna Levels ◽  
Es Cell ◽  
External Application ◽  
Self Renewal ◽  
Mouse Es Cells

Ion channels participate in cell homeostasis and are involved in the regulation of proliferation and differentiation in several cell types; however, their presence and function in embryonic stem (ES) cells are poorly studied. We have investigated the existence of voltage-dependent inward currents in mouse ES cells and their ability to modulate proliferation and self-renewal. Patch-clamped ES cells had inactivating tetrodotoxin (TTX)-sensitive Na+ currents as well as transient Ca2+ currents abolished by the external application of Ni2+. Biophysical and pharmacological data indicated that the Ca2+ current is predominantly mediated by T-type (Cav3.2) channels. The number of cells expressing T-type channels and Cav3.2 mRNA levels increased at the G1/S transition of the cell cycle. TTX had no effect on ES cell proliferation. However, blockade of T-type Ca2+ currents with Ni2+ induced a decrease in proliferation and alkaline phosphatase positive colonies as well as reduced expression of Oct3/4 and Nanog, all indicative of loss in self-renewal capacity. Decreased alkaline phosphatase and Oct3/4 expression were also observed in cells subjected to small interfering RNA-induced knockdown for T-type (Cav3.2) Ca2+ channels, thus partially recapitulating the pharmacological effects on self-renewal. These results indicate that Cav3.2 channel expression in ES cells is modulated along the cell cycle being induced at late G1 phase. They also suggest that these channels are involved in the maintenance of the undifferentiated state of mouse ES cells. We propose that Ca2+ entry mediated by Cav3.2 channels might be one of the intracellular signals that participate in the complex network responsible for ES cell self-renewal.

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.

scholarly journals Genesis of embryonic stem cells

2003 ◽  
Vol 358 (1436) ◽  
pp. 1397-1402 ◽  
Author(s):  
Mia Buehr ◽  
Austin Smith
Keyword(s):  
Kinase Inhibitor ◽  
Time Window ◽  
Es Cells ◽  
Primary Cultures ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Human Embryos ◽  
Es Cell

Embryonic stem (ES) cells are permanent pluripotent stem cell lines established from pre–implantation mouse embryos. There is currently great interest in the potential therapeutic applications of analogous cells derived from human embryos. The isolation of ES cells is commonly presented as a straightforward transfer of cells in the early embryo into culture. In reality, however, continuous expansion of pluripotent cells does not occur in vivo, and in vitro is the exception rather than the norm. Both genetic and epigenetic factors influence the ability to derive ES cells. We have tracked the expression of a key marker and determinant of pluripotency, the transcription factor Oct–4, in primary cultures of mouse epiblasts and used this to assay the effect of experimental manipulations on the maintenance of a pluripotent cell compartment. We find that expression of Oct–4 is often lost prior to overt cytodifferentiation of the epiblast. The rate and extent of Oct–4 extinction varies with genetic background. We report that treatment with the MAP kinase/ERK kinase inhibitor PD98059, which suppresses activation of the mitogen–activated protein kinases Erk1 and Erk2, results in increased persistence of Oct–4–expressing cells. Oct–4 expression is also relatively sustained in cultures of diapause embryos and of isolated inner cell masses. Combination of all three conditions allowed the derivation of germline–competent ES cells from the normally refractory CBA mouse strain. These findings suggest that the genesis of an ES cell is a relatively complex process requiring epigenetic modulation of key gene expression over a brief time–window. Procedures that extend this time–window and/or directly regulate the critical genes should increase the efficiency of ES cell derivation.

scholarly journals Embryonic stem cells commit to differentiation by symmetric divisions following a variable lag period

2020 ◽  
Author(s):  
Stanley E Strawbridge ◽  
Guy B Blanchard ◽  
Austin Smith ◽  
Hillel Kugler ◽  
Graziano Martello
Keyword(s):  
Cell Cycle ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Cell Identity ◽  
Daughter Cells ◽  
Population Dynamics Model ◽  
Developmental Progression ◽  
Lag Period

ABSTRACTMouse embryonic stem (ES) cells are derived from the epiblast of the preimplantation embryo and retain the capacity to give rise to all embryo lineages. ES cells can be released into differentiation from a near-homogeneous maintenance condition. Exit from the ES cell state can be accurately monitored using the Rex1-GFPd2 transgenic reporter, providing a powerful system for examining a mammalian cell fate transition. Here, we performed live-cell imaging and tracking of ES cells during entry into differentiation for 48 hours in defined conditions. We observed a greater cell surface area and a modest shortening of the cell cycle prior to exit and subsequently a reduction in cell size and increase in motility. We did not see any instance of cells regaining ES cell identity, consistent with unidirectional developmental progression. Transition occurred asynchronously across the population but genealogical tracking revealed a high correlation in cell-cycle length and Rex1-GFPd2 expression between daughter cells. A population dynamics model was consistent with symmetric divisions during exit from naive pluripotency. Collapse of ES cell identity occurred acutely in individual cells but after a variable delay. The variation in lag period can extend up to three generations, creating marked population asynchrony.

scholarly journals Serum Response Factor Is Required for Immediate-Early Gene Activation yet Is Dispensable for Proliferation of Embryonic Stem Cells

2001 ◽  
Vol 21 (8) ◽  
pp. 2933-2943 ◽  
Author(s):  
Gerhard Schratt ◽  
Birgit Weinhold ◽  
Ante S. Lundberg ◽  
Sebastian Schuck ◽  
Jürgen Berger ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Serum Response Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Gene ◽  
Response Factor ◽  
Es Cell ◽  
Cycle Progression ◽  
Serum Response

ABSTRACT Addition of serum to mitogen-starved cells activates the cellular immediate-early gene (IEG) response. Serum response factor (SRF) contributes to such mitogen-stimulated transcriptional induction of many IEGs during the G0-G1 cell cycle transition. SRF is also believed to be essential for cell cycle progression, as impairment of SRF activity by specific antisera or antisense RNA has previously been shown to block mammalian cell proliferation. In contrast, Srf −/− mouse embryos grow and develop up to E6.0. Using the embryonic stem (ES) cell system, we demonstrate here that wild-type ES cells do not undergo complete cell cycle arrest upon serum withdrawal but that they can mount an efficient IEG response. This IEG response, however, is severely impaired in Srf −/− ES cells, providing the first genetic proof that IEG activation is dependent upon SRF. Also, Srf−/− ES cells display altered cellular morphology, reduced cortical actin expression, and an impaired plating efficiency on gelatin. Yet, despite these defects, the proliferation rates of Srf −/− ES cells are not substantially altered, demonstrating that SRF function is not required for ES cell cycle progression.

scholarly journals Oct-4 controls cell-cycle progression of embryonic stem cells

2010 ◽  
Vol 426 (2) ◽  
pp. 171-181 ◽  
Author(s):  
Jungwoon Lee ◽  
Yeorim Go ◽  
Inyoung Kang ◽  
Yong-Mahn Han ◽  
Jungho Kim
Keyword(s):  
Cell Cycle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Cycle Progression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Functional Domains ◽  
Es Cell ◽  
Down Regulation ◽  
Cycle Progression

Mouse and human ES (embryonic stem) cells display unusual proliferative properties and can produce pluripotent stem cells indefinitely. Both processes might be important for maintaining the ‘stemness’ of ES cells; however, little is known about how the cell-cycle fate is regulated in ES cells. Oct-4, a master switch of pluripotency, plays an important role in maintaining the pluripotent state of ES cells and may prevent the expression of genes activated during differentiation. Using ZHBTc4 ES cells, we have investigated the effect of Oct-4 on ES cell-cycle control, and we found that Oct-4 down-regulation in ES cells inhibits proliferation by blocking cell-cycle progression in G0/G1. Deletion analysis of the functional domains of Oct-4 indicates that the overall integrity of the Oct-4 functional domains is important for the stimulation of S-phase entry. We also show in the present study that the p21 gene is a target for Oct-4 repression. Furthermore, p21 protein levels were repressed by Oct-4 and were induced by the down-regulation of Oct-4 in ZHBTc4 ES cells. Therefore the down-regulation of p21 by Oct-4 may contribute to the maintenance of ES cell proliferation.

scholarly journals Production of Mice Entirely Derived from Embryonic Stem (ES) Cell with Many Passages by Coculture of ES Cells with Cytochalasin B Induced Tetraploid Embryos.

1995 ◽  
Vol 44 (3) ◽  
pp. 205-210 ◽  
Author(s):  
Otoya UEDA ◽  
Kouichi JISHAGE ◽  
Nobuo KAMADA ◽  
Satomi UCHIDA ◽  
Hiroshi SUZUKI
Keyword(s):  
Cytochalasin B ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell

Germ line transmission of an inactive N-myc allele generated by homologous recombination in mouse embryonic stem cells

1990 ◽  
Vol 10 (12) ◽  
pp. 6755-6758
Author(s):  
B R Stanton ◽  
S W Reid ◽  
L F Parada
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Homologous Recombination ◽  
Embryonic Development ◽  
Cell Lines ◽  
Germ Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Es Cell ◽  
Germ Line Transmission

We have disrupted one allele of the N-myc locus in mouse embryonic stem (ES) cells by using homologous recombination techniques and have obtained germ line transmission of null N-myc ES cell lines with transmission of the null N-myc allele to the offspring. The creation of mice with a deficient N-myc allele will allow the generation of offspring bearing null N-myc alleles in both chromosomes and permit study of the role that this proto-oncogene plays in embryonic development.

Brachyury - a gene affecting mouse gastrulation and early organogenesis

Development ◽  
1992 ◽  
Vol 116 (Supplement) ◽  
pp. 157-165 ◽  
Author(s):  
R. S. P. Beddington ◽  
P. Rashbass ◽  
V. Wilson
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Primitive Streak ◽  
Coat Colour ◽  
Es Cell ◽  
Wild Type ◽  
Mesoderm Cell ◽  
Rostrocaudal Axis

Mouse embryos that are homozygous for the Brachyury (T) deletion die at mid-gestation. They have prominent defects in the notochord, the allantois and the primitive streak. Expression of the T gene commences at the onset of gastrulation and is restricted to the primitive streak, mesoderm emerging from the streak, the head process and the notochord. Genetic evidence has suggested that there may be an increasing demand for T gene function along the rostrocaudal axis. Experiments reported here indicate that this may not be the case. Instead, the gradient in severity of the T defect may be caused by defective mesoderm cell movements, which result in a progressive accumulation of mesoderm cells near the primitive streak. Embryonic stem (ES) cells which are homozygous for the T deletion have been isolated and their differentiation in vitro and in vivo compared with that of heterozygous and wild-type ES cell lines. In +/+ ↔ T/T ES cell chimeras the Brachyury phenotype is not rescued by the presence of wild-type cells and high level chimeras show most of the features characteristic of intact T/T mutants. A few offspring from blastocysts injected with T/T ES cells have been born, several of which had greatly reduced or abnormal tails. However, little or no ES cell contribution was detectable in these animals, either as coat colour pigmentation or by isozyme analysis. Inspection of potential +/+ ↔ T/T ES cell chimeras on the 11th or 12th day of gestation, stages later than that at which intact T/T mutants die, revealed the presence of chimeras with caudal defects. These chimeras displayed a gradient of ES cell colonisation along the rostrocaudal axis with increased colonisation of caudal regions. In addition, the extent of chimerism in ectodermal tissues (which do not invaginate during gastrulation) tended to be higher than that in mesodermal tissues (which are derived from cells invaginating through the primitive streak). These results suggest that nascent mesoderm cells lacking the T gene are compromised in their ability to move away from the primitive streak. This indicates that one function of the T genemay be to regulate cell adhesion or cell motility properties in mesoderm cells. Wild-type cells in +/+ ↔ T/T chimeras appear to move normally to populate trunk and head mesoderm, suggesting that the reduced motility in T/T cells is a cell autonomous defect

Human embryonic stem cells: challenges and opportunities

2006 ◽  
Vol 18 (8) ◽  
pp. 839 ◽  
Author(s):  
Steven L. Stice ◽  
Nolan L. Boyd ◽  
Sujoy K. Dhara ◽  
Brian A. Gerwe ◽  
David W. Machacek ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neural Stem Cells ◽  
Cell Line ◽  
Cell Fate ◽  
Neural Development ◽  
Es Cells ◽  
Embryonic Stem ◽  
Normal Karyotype ◽  
Es Cell ◽  
Cell Therapies

Human and non-human primate embryonic stem (ES) cells are invaluable resources for developmental studies, pharmaceutical research and a better understanding of human disease and replacement therapies. In 1998, subsequent to the establishment of the first monkey ES cell line in 1995, the first human ES cell line was developed. Later, three of the National Institute of Health (NIH) lines (BG01, BG02 and BG03) were derived from embryos that would have been discarded because of their poor quality. A major challenge to research in this area is maintaining the unique characteristics and a normal karyotype in the NIH-registered human ES cell lines. A normal karyotype can be maintained under certain culture conditions. In addition, a major goal in stem cell research is to direct ES cells towards a limited cell fate, with research progressing towards the derivation of a variety of cell types. We and others have built on findings in vertebrate (frog, chicken and mouse) neural development and from mouse ES cell research to derive neural stem cells from human ES cells. We have directed these derived human neural stem cells to differentiate into motoneurons using a combination of developmental cues (growth factors) that are spatially and temporally defined. These and other human ES cell derivatives will be used to screen new compounds and develop innovative cell therapies for degenerative diseases.

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