scholarly journals Bcl-2 inhibits retinoic acid-induced apoptosis during the neural differentiation of embryonal stem cells.

1996 ◽  
Vol 132 (5) ◽  
pp. 955-968 ◽  
Author(s):  
H Okazawa ◽  
J Shimizu ◽  
M Kamei ◽  
I Imafuku ◽  
H Hamada ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Dna Cleavage ◽  
Antisense Oligonucleotides ◽  
Neural Differentiation ◽  
Chromatin Condensation ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Apoptotic Cells ◽  
Endogenous Expression ◽  
Induced Apoptosis

We report here that all trans-retinoic acid (RA), a classical morphogen, induces apoptosis during the neural differentiation of the embryonic stem cell line P19. The apoptotic cells showed, in addition to DNA cleavage, typical morphological changes including chromatin condensation, nuclear fragmentation, and cytoplasmic vacuolation. These apoptotic changes became obvious by 12 h after the addition of RA. The endogenous expression of bcl-2 in surviving cells was down-regulated during this process, and the compelled expression of bcl-2 by retroviral vectors reduced the number of apoptotic cells. Apoptosis was partially inhibited by adding antisense oligonucleotides against RA receptors (RARs) simultaneously or by transfecting a plasmid vector flanked with a RA-responsive element. Antisense oligonucleotides against retinoid X receptors (RXRs), the receptors for 9 cis-RA, did not inhibit apoptosis induced by all trans-RA. Cycloheximide and actinomycin D, inhibitors of protein and RNA syntheses, respectively, suppressed apoptosis. No changes were seen in the expression of tumor necrosis factors, their receptors, Fas, FasL, p53, or c-myc, molecules which have been suggested to participate in the apoptotic process. Addition of neurotrophins to the culture medium did not affect apoptosis. These findings suggest that the signals themselves, promote expression of molecules essential for apoptosis. Furthermore, we observed that RA induced apoptosis of cerebral neurons from murine embryos in primary culture, which suggests that RA might participate in cell death which occurs during neural development.

scholarly journals A novel role for Gab2 in bFGF-mediated cell survival during retinoic acid–induced neuronal differentiation

2005 ◽  
Vol 170 (2) ◽  
pp. 305-316 ◽  
Author(s):  
Yingwei Mao ◽  
Angel W.-M. Lee
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Neuronal Differentiation ◽  
Embryonic Stem ◽  
Akt Activation ◽  
Important Player ◽  
Ec Cells ◽  
Phosphoinositide 3 Kinase ◽  
Induced Apoptosis ◽  
Constitutively Active

Gab proteins amplify and integrate signals stimulated by many growth factors. In culture and animals, retinoic acid (RA) induces neuronal differentiation. We show that Gab2 expression is detected in neurons in three models of neuronal differentiation: embryonic carcinoma (EC) stem cells, embryonic stem cells, and primary neural stem cells (NSCs). RA treatment induces apoptosis, countered by basic FGF (bFGF). In EC cells, Gab2 silencing results in hypersensitivity to RA-induced apoptosis and abrogates the protection by bFGF. Gab2 suppression reduces bFGF-dependent activation of AKT but not ERK, and constitutively active AKT, but not constitutively active MEK1, reverses the hypersensitization. Thus, Gab2-mediated AKT activation is required for bFGF's protection. Moreover, Gab2 silencing impairs the differentiation of EC cells to neurons. Similarly, in NSCs, Gab2 suppression reduces bFGF-dependent proliferation as well as neuronal survival and production upon differentiation. Our findings provide the first evidence that Gab2 is an important player in neural differentiation, partly by acting downstream of bFGF to mediate survival through phosphoinositide 3 kinase–AKT.

scholarly journals Selective apoptosis of pluripotent mouse and human stem cells by novel ceramide analogues prevents teratoma formation and enriches for neural precursors in ES cell–derived neural transplants

2004 ◽  
Vol 167 (4) ◽  
pp. 723-734 ◽  
Author(s):  
Erhard Bieberich ◽  
Jeane Silva ◽  
Guanghu Wang ◽  
Kannan Krishnamurthy ◽  
Brian G. Condie
Keyword(s):  
Stem Cell ◽  
Neural Differentiation ◽  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Human Stem Cells ◽  
Pluripotency Marker ◽  
Lineage Differentiation ◽  
Induced Apoptosis ◽  
Teratoma Formation

The formation of stem cell–derived tumors (teratomas) is observed when engrafting undifferentiated embryonic stem (ES) cells, embryoid body–derived cells (EBCs), or mammalian embryos and is a significant obstacle to stem cell therapy. We show that in tumors formed after engraftment of EBCs into mouse brain, expression of the pluripotency marker Oct-4 colocalized with that of prostate apoptosis response-4 (PAR-4), a protein mediating ceramide-induced apoptosis during neural differentiation of ES cells. We tested the ability of the novel ceramide analogue N-oleoyl serinol (S18) to eliminate mouse and human Oct-4(+)/PAR-4(+) cells and to increase the proportion of nestin(+) neuroprogenitors in EBC-derived cell cultures and grafts. S18-treated EBCs persisted in the hippocampal area and showed neuronal lineage differentiation as indicated by the expression of β-tubulin III. However, untreated cells formed numerous teratomas that contained derivatives of endoderm, mesoderm, and ectoderm. Our results show for the first time that ceramide-induced apoptosis eliminates residual, pluripotent EBCs, prevents teratoma formation, and enriches the EBCs for cells that undergo neural differentiation after transplantation.

Caspase-3 Activity and Carbonyl Cyanide m-Chlorophenylhydrazone-induced Apoptosis in HL-60 cells

2001 ◽  
Vol 29 (3) ◽  
pp. 243-249 ◽  
Author(s):  
Petr Mlejnek
Keyword(s):  
Cell Death ◽  
Dna Cleavage ◽  
Caspase 3 ◽  
Chromatin Condensation ◽  
Experimental System ◽  
Apoptotic Bodies ◽  
Nucleosomal Dna ◽  
Carbonyl Cyanide ◽  
Induced Apoptosis ◽  
Fluoromethyl Ketone

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being “truly” necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as “truly” necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.

scholarly journals A novel dual reporter embryonic stem cell line for toxicological assessment of teratogen-induced perturbation of anterior–posterior patterning of the heart

2019 ◽  
Vol 94 (2) ◽  
pp. 631-645 ◽  
Author(s):  
Robert S. Leigh ◽  
Heikki J. Ruskoaho ◽  
Bogac L. Kaynak
Keyword(s):  
Stem Cell ◽  
Retinoic Acid ◽  
Embryonic Stem Cell ◽  
Developmental Toxicity ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Reporter Expression ◽  
Dual Reporter ◽  
Anterior Posterior

AbstractReliable in vitro models to assess developmental toxicity of drugs and chemicals would lead to improvement in fetal safety and a reduced cost of drug development. The validated embryonic stem cell test (EST) uses cardiac differentiation of mouse embryonic stem cells (mESCs) to predict in vivo developmental toxicity, but does not take into account the stage-specific patterning of progenitor populations into anterior (ventricular) and posterior (atrial) compartments. In this study, we generated a novel dual reporter mESC line with fluorescent reporters under the control of anterior and posterior cardiac promoters. Reporter expression was observed in nascent compartments in transgenic mouse embryos, and mESCs were used to develop differentiation assays in which chemical modulators of Wnt (XAV939: 3, 10 µM), retinoic acid (all-trans retinoic acid: 0.1, 1, 10 µM; 9-cis retinoic acid: 0.1, 1, 10 µM; bexarotene 0.1, 1, 10 µM), and Tgf-β (SB431542: 3, 10 µM) pathways were tested for stage- and dose-dependent effects on in vitro anterior–posterior patterning. Our results suggest that with further development, the inclusion of anterior–posterior reporter expression could be part of a battery of high-throughput tests used to identify and characterize teratogens.

scholarly journals The ciliary proteins Meckelin and Jouberin are required for retinoic acid-dependent neural differentiation of mouse embryonic stem cells

2014 ◽  
Vol 87 (3-4) ◽  
pp. 134-146 ◽  
Author(s):  
Sveva Romani ◽  
Barbara Illi ◽  
Roberta De Mori ◽  
Mauro Savino ◽  
Joseph G. Gleeson ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Embryonic Stem Cells ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells

Interplay of leukemia inhibitory factor and retinoic acid on neural differentiation of mouse embryonic stem cells

2007 ◽  
Vol 85 (12) ◽  
pp. 2686-2701 ◽  
Author(s):  
Raquel Martín-Ibáñez ◽  
Noelia Urbán ◽  
Solène Sergent-Tanguy ◽  
José Ramón Pineda ◽  
Núria Garrido-Clua ◽  
...  
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Embryonic Stem Cells ◽  
Leukemia Inhibitory Factor ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cells ◽  
Inhibitory Factor
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