scholarly journals Disruption of the Talin Gene Compromises Focal Adhesion Assembly in Undifferentiated but Not Differentiated Embryonic Stem Cells

1998 ◽  
Vol 142 (4) ◽  
pp. 1121-1133 ◽  
Author(s):  
Helen Priddle ◽  
Lance Hemmings ◽  
Susan Monkley ◽  
Alison Woods ◽  
Bipin Patel ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Focal Adhesions ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Integrin Subunit ◽  
Β1 Integrin ◽  
Es Cell ◽  
Wild Type

We have used gene disruption to isolate two talin (−/−) ES cell mutants that contain no intact talin. The undifferentiated cells (a) were unable to spread on gelatin or laminin and grew as rounded colonies, although they were able to spread on fibronectin (b) showed reduced adhesion to laminin, but not fibronectin (c) expressed much reduced levels of β1 integrin, although levels of α5 and αV were wild-type (d) were less polarized with increased membrane protrusions compared with a vinculin (−/−) ES cell mutant (e) were unable to assemble vinculin or paxillin-containing focal adhesions or actin stress fibers on fibronectin, whereas vinculin (−/−) ES cells were able to assemble talin-containing focal adhesions. Both talin (−/−) ES cell mutants formed embryoid bodies, but differentiation was restricted to two morphologically distinct cell types. Interestingly, these differentiated talin (−/−) ES cells were able to spread and form focal adhesion-like structures containing vinculin and paxillin on fibronectin. Moreover, the levels of the β1 integrin subunit were comparable to those in wild-type ES cells. We conclude that talin is essential for β1 integrin expression and focal adhesion assembly in undifferentiated ES cells, but that a subset of differentiated cells are talin independent for both characteristics.

Lineage specific differentiation of pluripotent cells in vitro: a role for extraembryonic cell types

2001 ◽  
Vol 13 (1) ◽  
pp. 15 ◽  
Author(s):  
J. Rathjen ◽  
S. Dunn ◽  
M. D. Bettess ◽  
P. D. Rathjen
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Cell Aggregates ◽  
Visceral Endoderm ◽  
Es Cell ◽  
Pluripotent Cells ◽  
Developmental Potential

The controlled differentiation of pluripotent cells will be a prerequisite for many cell therapies. We have previously reported homogeneous conversion of embryonic stem (ES) cells in vitro to early primitive ectoderm-like (EPL) cells, equivalent to early primitive ectoderm, an obligatory differentiation intermediate between ES cells and somatic cell populations. Early primitive ectoderm-like cells differentiated within aggregates form mesodermal lineages at the expense of ectoderm. In this work we demonstrate that the failure of EPL cells to form ectodermal cell types does not reflect an inherent restriction in developmental potential. Early primitive ectoderm-like cells form ectodermal derivatives such as neurons in response to neural inducers such as retinoic acid, or when differentiated in the environment provided by ES cell embryoid bodies. This could be explained by signals from the extraembryonic cell type visceral endoderm which forms in differentiating ES cell but not EPL cell aggregates. Consistent with this possibility, culture of EPL cell aggregates in the presence of visceral endoderm-like signals did not prevent differentiation of the pluripotent cells, but resulted in suppression of mesoderm formation. These results suggest a role for visceral endoderm in regulation of germ layer specification from pluripotent cells, and can be integrated into a model for cell differentiation in vitro and in vivo.

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

scholarly journals Designer blood: creating hematopoietic lineages from embryonic stem cells

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1265-1275 ◽  
Author(s):  
Abby L. Olsen ◽  
David L. Stachura ◽  
Mitchell J. Weiss
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Basic Research ◽  
Cell Types ◽  
Patient Specific ◽  
Es Cell ◽  
Blood Disorders ◽  
Hematopoietic Stem

Embryonic stem (ES) cells exhibit the remarkable capacity to become virtually any differentiated tissue upon appropriate manipulation in culture, a property that has been beneficial for studies of hematopoiesis. Until recently, the majority of this work used murine ES cells for basic research to elucidate fundamental properties of blood-cell development and establish methods to derive specific mature lineages. Now, the advent of human ES cells sets the stage for more applied pursuits to generate transplantable cells for treating blood disorders. Current efforts are directed toward adapting in vitro hematopoietic differentiation methods developed for murine ES cells to human lines, identifying the key interspecies differences in biologic properties of ES cells, and generating ES cell-derived hematopoietic stem cells that are competent to repopulate adult hosts. The ultimate medical goal is to create patient-specific and generic ES cell lines that can be expanded in vitro, genetically altered, and differentiated into cell types that can be used to treat hematopoietic diseases.

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.

Development to term of mouse androgenetic aggregation chimeras

Development ◽  
1991 ◽  
Vol 113 (4) ◽  
pp. 1325-1333 ◽  
Author(s):  
J.R. Mann ◽  
C.L. Stewart
Keyword(s):  
Mouse Strain ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Considerable Improvement ◽  
Es Cell ◽  
Partial Explanation ◽  
Developmental Potential ◽  
Skeletal Abnormalities ◽  
Embryonic Tissues

Diploid androgenetic eggs contain two sperm-derived genomes, and only rarely develop to the early somite stage. Also, previous studies have indicated that androgenetic eggs cannot be rescued in aggregation chimeras beyond embryonic stages. Paradoxically, in blastocyst injection chimeras made with androgenetic embryonic stem (ES) cells of the 129/Sv strain, we previously obtained considerable improvement in developmental potential. Although considerable death occurred in utero, overtly normal chimeric fetuses and occasional postnatal chimeras that developed skeletal abnormalities were observed. Consequently, we have re-evaluated the developmental potential of androgenetic aggregation chimeras utilizing androgenetic eggs of the 129/Sv strain, and of the BALB/c and CD-1 strains for comparison. Regardless of strain, androgenetic aggregation chimeras were generally more inviable than previously observed with androgenetic ES cell chimeras, and often the embryoproper was abnormal even when an androgenetic contribution was detected only in the extra-embryonic membranes. This is at least a partial explanation of the greater viability of androgenetic ES cell chimeras, as ES cells do not colonize significantly certain extra-embryonic tissues. Nevertheless, in the 129/Sv strain, occasional development of chimeras to term was obtained, and one chimera that survived postnatally developed identical skeletal abnormalities to those observed previously in androgenetic ES cell chimeras. This result demonstrates that at least one example of paternal imprinting is faithfully conserved in androgenetic ES cells. Also, the postnatal chimerism shows that androgenetic eggs can give rise to terminally differentiated cell types, and are therefore pluripotent. In contrast, only possibly one BALB/c and no CD-1 androgenetic aggregation chimeras developed to term. Therefore, the developmental potential of androgenetic aggregation chimeras is to some extent dependent on mouse strain.

scholarly journals A Shortened Life Span of EKLF−/− Adult Erythrocytes, Due to a Deficiency of β-Globin Chains, Is Ameliorated by Human γ-Globin Chains

Blood ◽  
1997 ◽  
Vol 90 (3) ◽  
pp. 1291-1299 ◽  
Author(s):  
Sai-Kiang Lim ◽  
James J. Bieker ◽  
Chyuan-Sheng Lin ◽  
Frank Costantini
Keyword(s):  
Life Span ◽  
Es Cells ◽  
Globin Gene ◽  
Embryonic Stem ◽  
Es Cell ◽  
Wild Type ◽  
Rt Pcr ◽  
Mature Erythrocyte ◽  
Heinz Bodies ◽  
Globin Chains

Abstract Using homologous recombination, both EKLF alleles in murine embryonic stem (ES) cells were inactivated. These EKLF−/− ES cells were capable of undergoing in vitro differentiation to form definitive erythroid colonies that were similar in size and number to those formed by wild-type ES cells. However, the EKLF−/− colonies were poorly hemoglobinized and enucleated erythrocytes in these colonies contained numerous Heinz bodies. Reverse transcriptase-polymerase chain reaction (RT-PCR) analyses revealed that adult and embryonic globin genes were appropriately regulated, with the exception of βh1-globin, which continued to be expressed at a very low level. The ratio of adult β-globin/α-globin mRNA in the mutant ES cells was 1/15 of that in wild-type ES cells. When the EKLF−/− cells were injected into blastocysts, they did not contribute at a detectable level to the mature erythrocyte compartment of the chimeric animals, based on analysis of glucose phosphate isomerase-1 (GPI-1) isozymes and hemoglobins that distinguish ES cell-derived erythrocytes from host blastocyst-derived erythrocytes. In contrast, semiquantitative RT-PCR analysis of RNA from reticulocytes of the same chimeric animals suggested that the ES cell-derived reticulocytes were present at a level of 6% to 8%. This indicated that the EKLF−/− erythrocytes in adult animals must be short-lived, apparently due to the imbalance of β-versus α-globin chains, leading to the precipitation of excess α-globin chains to form Heinz bodies. Consistent with this hypothesis, the short life span was ameliorated by introduction into the EKLF−/− ES cells of a human LCR/γ-globin gene, as evidenced by the presence of ES cell-derived reticulocytes as well as mature erythrocytes in the blood of the chimeric animals.

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.

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 Serum response factor is crucial for actin cytoskeletal organization and focal adhesion assembly in embryonic stem cells

2002 ◽  
Vol 156 (4) ◽  
pp. 737-750 ◽  
Author(s):  
Gerhard Schratt ◽  
Ulrike Philippar ◽  
Jürgen Berger ◽  
Heinz Schwarz ◽  
Olaf Heidenreich ◽  
...  
Keyword(s):  
Focal Adhesion ◽  
Serum Response Factor ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Spreading ◽  
Stress Fibers ◽  
Response Factor ◽  
Es Cell ◽  
And Migration ◽  
Serum Response

The activity of serum response factor (SRF), an essential transcription factor in mouse gastrulation, is regulated by changes in actin dynamics. Using Srf(−/−) embryonic stem (ES) cells, we demonstrate that SRF deficiency causes impairments in ES cell spreading, adhesion, and migration. These defects correlate with defective formation of cytoskeletal structures, namely actin stress fibers and focal adhesion (FA) plaques. The FA proteins FA kinase (FAK), β1-integrin, talin, zyxin, and vinculin were downregulated and/or mislocalized in ES cells lacking SRF, leading to inefficient activation of the FA signaling kinase FAK. Reduced overall actin expression levels in Srf(−/−) ES cells were accompanied by an offset treadmilling equilibrium, resulting in lowered F-actin levels. Expression of active RhoA-V14 rescued F-actin synthesis but not stress fiber formation. Introduction of constitutively active SRF-VP16 into Srf(−/−) ES cells, on the other hand, strongly induced expression of FA components and F-actin synthesis, leading to a dramatic reorganization of actin filaments into stress fibers and lamellipodia. Thus, using ES cell genetics, we demonstrate for the first time the importance of SRF for the formation of actin-directed cytoskeletal structures that determine cell spreading, adhesion, and migration. Our findings suggest an involvement of SRF in cell migratory processes in multicellular organisms.

scholarly journals Tumor Suppression by a Severely Truncated Species of Retinoblastoma Protein

2002 ◽  
Vol 22 (9) ◽  
pp. 3103-3110 ◽  
Author(s):  
Hong Yang ◽  
Bart O. Williams ◽  
Phillip W. Hinds ◽  
T. Shane Shih ◽  
Tyler Jacks ◽  
...  
Keyword(s):  
Tumor Suppression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Retinoblastoma Protein ◽  
Pituitary Tumors ◽  
Es Cell ◽  
Chimeric Mice ◽  
Wild Type ◽  
Substantial Fraction

ABSTRACT Rb+/+:Rb−/− chimeric mice are healthy until early in adulthood when they develop lethal pituitary tumors composed solely of Rb−/− cells. In an effort to delineate the minimal structures of the retinoblastoma protein necessary for RB tumor suppression function, chimeric animals derived from stably transfected RB−/− embryonic stem (ES) cells were generated. One such ES cell transfectant expressed a human RB allele encoding a stable, truncated nuclear derivative lacking residues 1 to 378 (Δ1-378). Others encoded either wild-type human RB or an internally deleted derivative of the Δ1-378 mutant. All gave rise to viable chimeric animals with comparable degrees of chimerism. However, unlike control mice derived, in part, from naive Rb−/− ES cells or from ES cells transformed by the double RB mutant, Δ1-378/Δexon22, animals derived from either wild-type RB- or Δ1-378 RB-producing ES cells failed to develop pituitary tumors. Thus, in this setting, a substantial fraction of the RB sequence is unnecessary for RB-mediated tumor suppression.

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