scholarly journals High-Content Screening for Chemical Modulators of Embryonal Carcinoma Cell Differentiation and Survival

2011 ◽  
Vol 16 (6) ◽  
pp. 603-617 ◽  
Author(s):  
Ivana Barbaric ◽  
Mark Jones ◽  
David J. Harley ◽  
Paul J. Gokhale ◽  
Peter W. Andrews
Keyword(s):  
Stem Cell ◽  
Cell Survival ◽  
Cell Fate ◽  
Embryonal Carcinoma ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryonal Carcinoma Cell ◽  
Screening Assay ◽  
Self Renewal ◽  
Formidable Challenge

Disentangling the complex interactions that govern stem cell fate choices of self-renewal, differentiation, or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that improved cell survival or induced differentiation. The survival compounds Y-27632, HA-1077, and H-8 all strongly inhibit the kinases ROCK and PRK2, highlighting the important role of these kinases in EC cell survival. Two molecules, GF109203x and rottlerin, induced EC differentiation. The effects of rottlerin were also investigated in human ES cells. Rottlerin inhibited the self-renewal ability of ES cells, caused the cell cycle arrest, and repressed the expression of pluripotency-associated genes.

scholarly journals Interaction of retinoic acid and scl controls primitive blood development

Blood ◽  
2010 ◽  
Vol 116 (2) ◽  
pp. 201-209 ◽  
Author(s):  
Jill L. O. de Jong ◽  
Alan J. Davidson ◽  
Yuan Wang ◽  
James Palis ◽  
Praise Opara ◽  
...  
Keyword(s):  
Stem Cell ◽  
Retinoic Acid ◽  
Cell Fate ◽  
Embryonic Stem ◽  
Yolk Sac ◽  
Erythroid Progenitors ◽  
Self Renewal ◽  
Hematopoietic Development ◽  
Blood Island ◽  
Primitive Hematopoiesis

Abstract Hematopoietic development during embryogenesis involves the interaction of extrinsic signaling pathways coupled to an intrinsic cell fate that is regulated by cell-specific transcription factors. Retinoic acid (RA) has been linked to stem cell self-renewal in adults and also participates in yolk sac blood island formation. Here, we demonstrate that RA decreases gata1 expression and blocks primitive hematopoiesis in zebrafish (Danio rerio) embryos, while increasing expression of the vascular marker, fli1. Treatment with an inhibitor of RA biosynthesis or a retinoic acid receptor antagonist increases gata1+ erythroid progenitors in the posterior mesoderm of wild-type embryos and anemic cdx4−/− mutants, indicating a link between the cdx-hox signaling pathway and RA. Overexpression of scl, a DNA binding protein necessary for hematopoietic development, rescues the block of hematopoiesis induced by RA. We show that these effects of RA and RA pathway inhibitors are conserved during primitive hematopoiesis in murine yolk sac explant cultures and embryonic stem cell assays. Taken together, these data indicate that RA inhibits the commitment of mesodermal cells to hematopoietic fates, functioning downstream of cdx4 and upstream of scl. Our studies establish a new connection between RA and scl during development that may participate in stem cell self-renewal and hematopoietic differentiation.

scholarly journals c-Jun NH2-Terminal Kinase Is Required for Lineage-Specific Differentiation but Not Stem Cell Self-Renewal

2010 ◽  
Vol 30 (6) ◽  
pp. 1329-1340 ◽  
Author(s):  
Ping Xu ◽  
Roger J. Davis
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Wild Type ◽  
Self Renewal ◽  
Chemical Genetic ◽  
Specific Differentiation ◽  
Gene Ablation ◽  
Early Embryonic Lethality

ABSTRACT The c-Jun NH2-terminal kinase (JNK) is implicated in proliferation. Mice with a deficiency of either the Jnk1 or the Jnk2 genes are viable, but a compound deficiency of both Jnk1 and Jnk2 causes early embryonic lethality. Studies using conditional gene ablation and chemical genetic approaches demonstrate that the combined loss of JNK1 and JNK2 protein kinase function results in rapid senescence. To test whether this role of JNK was required for stem cell proliferation, we isolated embryonic stem (ES) cells from wild-type and JNK-deficient mice. We found that Jnk1 −/− Jnk2 −/− ES cells underwent self-renewal, but these cells proliferated more rapidly than wild-type ES cells and exhibited major defects in lineage-specific differentiation. Together, these data demonstrate that JNK is not required for proliferation or self-renewal of ES cells, but JNK plays a key role in the differentiation of ES cells.

scholarly journals The Role of SMAD4 in Human Embryonic Stem Cell Self-Renewal and Stem Cell Fate

Stem Cells ◽  
2010 ◽  
pp. N/A-N/A ◽  
Author(s):  
Stuart Avery ◽  
Gaetano Zafarana ◽  
Paul J. Gokhale ◽  
Peter W. Andrews
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Fate ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Stem Cell Fate

scholarly journals Stem cell culture on polyvinyl alcohol hydrogels having different elasticity and immobilized with ECM-derived oligopeptides

2017 ◽  
Vol 37 (7) ◽  
pp. 647-660 ◽  
Author(s):  
Saradaprasan Muduli ◽  
Li-Hua Chen ◽  
Meng-Pei Li ◽  
Zhao-wen Heish ◽  
Cheng-Hui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Culture ◽  
Extracellular Matrix ◽  
Stem Cell ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Poly Vinyl Alcohol ◽  
Hematopoietic Stem ◽  
Stem Cell Culture

Abstract The physical characteristics of cell culture materials, such as their elasticity, affect stem cell fate with respect to cell proliferation and differentiation. We systematically investigated the morphologies and characteristics of several stem cell types, including human amniotic-derived stem cells, human hematopoietic stem cells, human induced pluripotent stem (iPS) cells, and embryonic stem (ES) cells on poly(vinyl alcohol) (PVA) hydrogels immobilized with and without extracellular matrix-derived oligopeptide. Human ES cells did not adhere well to soft PVA hydrogels immobilized with oligovitronectin, whereas they did adhere well to PVA hydrogel dishes with elasticities greater than 15 kPa. These results indicate that biomaterials such as PVA hydrogels should be designed to possess minimum elasticity to facilitate human ES cell attachment. PVA hydrogels immobilized with and without extracellular matrix-derived oligopeptides are excellent candidates of cell culture biomaterials for investigations into how cell culture biomaterial elasticity affects stem cell culture and differentiation.

scholarly journals Embryonic stem cells assume a primitive neural stem cell fate in the absence of extrinsic influences

2006 ◽  
Vol 172 (1) ◽  
pp. 79-90 ◽  
Author(s):  
Simon R. Smukler ◽  
Susan B. Runciman ◽  
Shunbin Xu ◽  
Derek van der Kooy
Keyword(s):  
Stem Cell ◽  
Cell Fate ◽  
Neural Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Neural Cells ◽  
Neural Fate ◽  
Default State ◽  
Genetic Interference ◽  
Will Self

The mechanisms governing the emergence of the earliest mammalian neural cells during development remain incompletely characterized. A default mechanism has been suggested to underlie neural fate acquisition; however, an instructive process has also been proposed. We used mouse embryonic stem (ES) cells to explore the fundamental issue of how an uncommitted, pluripotent mammalian cell will self-organize in the absence of extrinsic signals and what cellular fate will result. To assess this default state, ES cells were placed in conditions that minimize external influences. Individual ES cells were found to rapidly transition directly into neural cells, a process shown to be independent of suggested instructive factors (e.g., fibroblast growth factors). Further, we provide evidence that the default neural identity is that of a primitive neural stem cell (NSC). The exiguous conditions used to reveal the default state were found to present primitive NSCs with a survival challenge (limiting their persistence and proliferation), which could be mitigated by survival factors or genetic interference with apoptosis.

Transcriptional profiling of reporter genes used for molecular imaging of embryonic stem cell transplantation

2006 ◽  
Vol 25 (1) ◽  
pp. 29-38 ◽  
Author(s):  
Joseph C. Wu ◽  
Joshua M. Spin ◽  
Feng Cao ◽  
Shuan Lin ◽  
Xiaoyan Xie ◽  
...  
Keyword(s):  
Stem Cell ◽  
Molecular Imaging ◽  
Cell Fate ◽  
Reporter Gene ◽  
Lentiviral Vector ◽  
Es Cells ◽  
Embryonic Stem ◽  
Reporter Genes ◽  
Nucleic Acid Metabolism ◽  
Es Cell

Stem cell therapy offers exciting promise for treatment of ischemic heart disease. Recent advances in molecular imaging techniques now allow investigators to monitor cell fate noninvasively and repetitively. Here we examine the effects of a triple-fusion reporter gene on embryonic stem (ES) cell transcriptional profiles. Murine ES cells were stably transfected with a self-inactivating lentiviral vector carrying a triple-fusion (TF) construct consisting of fluorescence, bioluminescence, and positron emission tomography (PET) reporter genes. Fluorescence-activated cell sorting (FACS) analysis allowed isolation of stably transfected populations. Microarray studies comparing gene expression in nontransduced control ES cells vs. stably transduced ES cells expressing triple fusion (ES-TF) revealed some increases in transcriptional variability. Annotation analysis showed that ES-TF cells downregulated cell cycling, cell death, and protein and nucleic acid metabolism genes while upregulating homeostatic and anti-apoptosis genes. Despite these transcriptional changes, expression of the TF reporter gene had no significant effects on ES cell viability, proliferation, and differentiation capability. Importantly, transplantation studies in murine myocardium demonstrated the feasibility of tracking ES-TF cells in living subjects using bioluminescence and PET imaging. Taken together, this is the first study to analyze in detail the effects of reporter genes on molecular imaging of ES cells.

scholarly journals Distinct SoxB1 networks are required for naïve and primed pluripotency

2017 ◽  
Author(s):  
Andrea Corsinotti ◽  
Frederick C. K. Wong ◽  
Tülin Tatar ◽  
Iwona Szczerbinska ◽  
Florian Halbritter ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Neural Differentiation ◽  
Embryonic Stem ◽  
Functional Redundancy ◽  
Self Renewal ◽  
Cell Fate Decisions ◽  
Pluripotent State

AbstractDeletion of Sox2 from embryonic stem cells (ESCs) causes trophectodermal differentiation. While this can be prevented by enforced expression of the related SOXB1 proteins, SOX1 or SOX3, the roles of SOXB1 proteins in epiblast stem cell (EpiSC) pluripotency are unknown. Here we show that Sox2 can be deleted from EpiSCs with impunity. This is due to a shift in the balance of SoxB1 expression in EpiSCs, which have decreased Sox2 and increased Sox3 compared to ESCs. Consistent with functional redundancy, Sox3 can also be deleted from EpiSCs without eliminating self-renewal. However, deletion of both Sox2 and Sox3 prevents self-renewal. The overall SOXB1 levels in ESCs affect differentiation choices: neural differentiation of Sox2 heterozygous ESCs is compromised, while increased SOXB1 levels divert the ESC to EpiSC transition towards neural differentiation. Therefore, optimal SOXB1 levels are critical for each pluripotent state and for cell fate decisions during exit from naïve pluripotency.

Sign in / Sign up

Export Citation Format

Share Document