scholarly journals Phenotypic Complementation Establishes Requirements for Specific POU Domain and Generic Transactivation Function of Oct-3/4 in Embryonic Stem Cells

2002 ◽  
Vol 22 (5) ◽  
pp. 1526-1536 ◽  
Author(s):  
Hitoshi Niwa ◽  
Shinji Masui ◽  
Ian Chambers ◽  
Austin G. Smith ◽  
Jun-ichi Miyazaki
Keyword(s):  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Phenotype ◽  
Dependent Manner ◽  
Transactivation Domain ◽  
Es Cell ◽  
Self Renewal ◽  
Pou Domain ◽  
Cell Propagation

ABSTRACT Transcription factors of the POU family govern cell fate through combinatorial interactions with coactivators and corepressors. The POU factor Oct-3/4 can define differentiation, dedifferentation, or self-renewal of pluripotent embryonic stem (ES) cells in a sensitive, dose-dependent manner (H. Niwa, J.-I. Miyazali, and A. G. Smith, Nat. Genet. 24:372-376, 2000). Here we have developed a complementation assay based on the ability of Oct-3/4 transgenes to rescue self-renewal in conditionally null ES cells and used this to define which domains of Oct-3/4 are required to sustain the undifferentiated stem cell phenotype. Surprisingly, we found that molecules lacking either the N-terminal or C-terminal transactivation domain, though not both, can effectively replace full-length Oct-3/4. Furthermore, a fusion of the heterologous transactivation domain of Oct-2 to the Oct-3/4 POU domain can also sustain self-renewal. Thus, the unique function of Oct-3/4 in ES cell propagation resides in combination of the specific POU domain with a generic proline-rich transactivation domain. Interestingly, however, Oct-3/4 target gene expression elicited by the N- and C-terminal transactivation domains is not identical, indicating that at least one class of genes activated by Oct-3/4 is not required for ES cell propagation.

scholarly journals StemBond hydrogels optimise the mechanical microenvironment for embryonic stem cells

2019 ◽  
Author(s):  
Céline Labouesse ◽  
Chibeza C. Agley ◽  
Bao Xiu Tan ◽  
Moritz Hofer ◽  
Alex Winkel ◽  
...  
Keyword(s):  
Cell Fate ◽  
Cell Attachment ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cellular Reprogramming ◽  
Substrate Stiffness ◽  
Cell Fate Specification ◽  
Es Cell ◽  
Self Renewal ◽  
Mechanical Microenvironment

ABSTRACTStudies of mechanical signalling are typically performed by comparing cells cultured on soft and stiff hydrogel-based substrates. However, it is challenging to independently and robustly control both substrate stiffness and tethering of extracellular matrix (ECM) to substrates, making ECM tethering a potentially confounding variable in mechanical signalling investigations. Moreover, poor ECM tethering can lead to weak cell attachment. To address this, we developed StemBond hydrogels, a hydrogel formulation in which ECM tethering is stable and can be varied independently of stiffness. We show that soft StemBond hydrogels provide an optimal format for culturing embryonic stem (ES) cells. We find that soft StemBond substrates improve the homogeneity of ES cell populations, boost their self-renewal, and increase the efficiency of cellular reprogramming. Our findings underline how soft microenvironments impact mechanosensitive signalling pathways regulating self-renewal and differentiation, indicating that optimising the complete mechanical microenvironment will offer greater control over stem cell fate specification.

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.

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.

The WNT receptor FZD7 contributes to self-renewal signaling of human embryonic stem cells

2008 ◽  
Vol 389 (7) ◽  
Author(s):  
Kai Melchior ◽  
Jonathan Weiß ◽  
Holm Zaehres ◽  
Yong-mi Kim ◽  
Carolyn Lutzko ◽  
...  
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Marker Genes ◽  
Specific Marker ◽  
Mrna Levels ◽  
Es Cell ◽  
Specific Expression ◽  
Self Renewal ◽  
Human Es Cells

Abstract A number of recent studies identified nuclear factors that together have the unique ability to induce pluripotency in differentiated cell types. However, little is known about the factors that are needed to maintain human embryonic stem (ES) cells in an undifferentiated state. In a search for such requirements, we performed a comprehensive meta-analysis of publicly available SAGE and microarray data. The rationale for this analysis was to identify genes that are exclusively expressed in human ES cell lines compared to 30 differentiated tissue types. The WNT receptor FZD7 was found among the genes with an ES cell-specific expression profile in both SAGE and microarray analyses. Subsequent validation by quantitative RT-PCR and flow cytometry confirmed that FZD7 mRNA levels in human ES cells are up to 200-fold higher compared to differentiated cell types. ShRNA-mediated knockdown of FZD7 in human ES cells induced dramatic changes in the morphology of ES cell colonies, perturbation of expression levels of germ layer-specific marker genes, and a rapid loss of expression of the ES cell-specific transcription factor OCT4. These findings identify the WNT receptor FZD7 as a novel ES cell-specific surface antigen with a likely important role in the maintenance of ES cell self-renewal capacity.

scholarly journals Endogenous fluctuations of OCT4 and SOX2 bias pluripotent cell fate decisions

2018 ◽  
Author(s):  
Daniel Strebinger ◽  
Cédric Deluz ◽  
Elias T. Friman ◽  
Subashika Govindan ◽  
Andrea B. Alber ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cell Fate ◽  
Es Cells ◽  
Embryonic Stem ◽  
Chromatin Accessibility ◽  
Directed Differentiation ◽  
Es Cell ◽  
Endogenous Fluctuations ◽  
Cell Fate Decisions ◽  
Oct4 Protein

AbstractSOX2 and OCT4 are pioneer transcription factors playing a key role in embryonic stem (ES) cell self-renewal and differentiation. However, how temporal fluctuations in their expression levels bias lineage commitment is unknown. Here we generated knock-in reporter fusion ES cell lines allowing to monitor endogenous SOX2 and OCT4 protein fluctuations in living cells and to determine their impact on mesendodermal and neuroectodermal commitment. We found that small differences in SOX2 and OCT4 levels impact cell fate commitment in G1 but not in S phase. Elevated SOX2 levels modestly increased neuroectodermal commitment and decreased mesendodermal commitment upon directed differentiation. In contrast, elevated OCT4 levels strongly biased ES cell towards both neuroectodermal and mesendodermal fates. Using ATAC-seq on ES cells gated for different endogenous SOX2 and OCT4 levels, we found that high OCT4 levels increased chromatin accessibility at differentiation-associated enhancers. This suggests that small endogenous fluctuations of pioneer transcription factors can bias cell fate decisions by concentration-dependent priming of differentiation-associated enhancers.

scholarly journals H4K20me3 methyltransferase SUV420H2 shapes the chromatin landscape of pluripotent embryonic stem cells

Development ◽  
2020 ◽  
Vol 147 (23) ◽  
pp. dev188516
Author(s):  
Jiji T. Kurup ◽  
Zhijun Han ◽  
Wenfei Jin ◽  
Benjamin L. Kidder
Keyword(s):  
Gene Expression ◽  
Cell Fate ◽  
Es Cells ◽  
Three Dimensional ◽  
Embryonic Stem ◽  
Pericentric Heterochromatin ◽  
Es Cell ◽  
Chromatin Interactions ◽  
Repressive Histone Modification ◽  
Dna Elements

ABSTRACTHeterochromatin, a densely packed chromatin state that is transcriptionally silent, is a critical regulator of gene expression. However, it is unclear how the repressive histone modification H4K20me3 or the histone methyltransferase SUV420H2 regulates embryonic stem (ES) cell fate by patterning the epigenetic landscape. Here, we report that depletion of SUV420H2 leads to a near-complete loss of H4K20me3 genome wide, dysregulated gene expression and delayed ES cell differentiation. SUV420H2-bound regions are enriched with repetitive DNA elements, which are de-repressed in SUV420H2 knockout ES cells. Moreover, SUV420H2 regulation of H4K20me3-marked heterochromatin controls chromatin architecture, including fine-scale chromatin interactions in pluripotent ES cells. Our results indicate that SUV420H2 plays a crucial role in stabilizing the three-dimensional chromatin landscape of ES cells, as loss of SUV420H2 resulted in A/B compartment switching, perturbed chromatin insulation, and altered chromatin interactions of pericentric heterochromatin and surrounding regions, indicative of localized decondensation. In addition, depletion of SUV420H2 resulted in compromised interactions between H4K20me3 and gene-regulatory regions. Together, these findings describe a new role for SUV420H2 in regulating the chromatin landscape of ES cells.

Efficient Non-Viral Transfection of Mouse and Human Embryonic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 5267-5267
Author(s):  
Zwi N. Berneman ◽  
Jeremy P. Brown ◽  
Sjaak Van der Sar ◽  
Dave Van den Plas ◽  
Lena Van den Eeden ◽  
...  
Keyword(s):  
Cell Lines ◽  
Es Cells ◽  
Embryonic Stem ◽  
Reporter Genes ◽  
Cell Phenotype ◽  
Es Cell ◽  
Short Term ◽  
Human Es Cells ◽  
Mrna Electroporation ◽  
Mouse Es Cell

Abstract BACKGROUND: Development of efficient non-viral gene transfer technologies for embryonic stem (ES) cells is urgently needed for various existing and new ES cell-based research strategies. In this study we investigated mRNA electroporation as a tool for short-term gene transfer in both mouse and human ES cells. METHODS: Culture and mRNA electroporation conditions for feeder-free cultured mouse and human ES cells were optimized on three mouse ES cell lines (E14, R1 and HM-1) and one human ES cell line (H9). After electroporation with EGFP mRNA, transfected ES cell populations were analyzed by FACS for EGFP expression, viability and phenotype. Also, stably-transfected mouse ES cell lines containing Lox-P or FRT-flanked reporter genes were electroporated with mRNA encoding Cre- or FLPe-recombinase proteins. Monitoring recombination efficiency was done based on the appearance and/or disappearance of fluorescent reporter genes, as determined by FACS analysis. ES cells that underwent recombination were further analyzed for potential to differentiate towards the neural lineage and differentiated cells were analyzed by FACS for expression of neural markers. RESULTS: (A) Electroporation of EGFP mRNA in mouse ES cells resulted in high level transgene expression (>90% EGFP positive cells) combined with low electroporation-induced cell mortality (>90% viable cells). Moreover, the electroporation procedure did not influence ES cell phenotype and further cell culture of undifferentiated ES cell populations. Electroporation of mRNA encoding Cre- or FLPe-recombinase proteins in stably-transfected mouse ES cell lines containing LoxP- or FRT-flanked reporter genes resulted in a recombination efficiency of respectively 75% and 90%. Moreover, these recombination events did not have influence on ES cell phenotype, viability, growth potential, and their ability to differentiate towards neural cell types upon retinoic acid stimulation. (B) Although human ES cells are much more sensitive as compared to mouse ES cells, we were able to develop improved culture and electroporation conditions for feeder-free maintained H9 human ES cells, which resulted in high level transgene expression (>90% EGFP+ cells) combined with high cell viability (>90% viable cells) after EGFP mRNA electroporation. CONCLUSIONS: RNA electroporation is a highly efficient method for short-term genetic loading of both mouse and human ES cells. Ongoing research now focuses on either short-term (via direct mRNA electroporation) or sustained (via mRNA-based FLPe-recombination) expression of transcription factors in ES cells and their influence on cell-fate within in vitro cultured embryoid bodies.

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 Effects of Synthetic Neural Adhesion Molecule Mimetic Peptides and Related Proteins on the Cardiomyogenic Differentiation of Mouse Embryonic Stem Cells

2015 ◽  
Vol 35 (6) ◽  
pp. 2437-2450 ◽  
Author(s):  
Ruodan Xu ◽  
Sureshkumar Perumal Srinivasan ◽  
Poornima Sureshkumar ◽  
Erastus Nembu Nembo ◽  
Christoph Schäfer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Synthetic Peptides ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dependent Manner ◽  
Es Cell ◽  
Cardiomyogenic Differentiation ◽  
Cellular Environment ◽  
Structural And Functional Properties

Background/Aims: Pluripotent stem cells differentiating into cardiomyocyte-like cells in an appropriate cellular environment have attracted significant attention, given the potential use of such cells for regenerative medicine. However, the precise mechanisms of lineage specification of pluripotent stem cells are still largely to be explored. Identifying the role of various small synthetic peptides involved in cardiomyogenesis may provide new insights into pathways promoting cardiomyogenesis. Methods: In the present study, using a transgenic murine embryonic stem (ES) cell lineage expressing enhanced green fluorescent protein (EGFP) under the control of α-myosin heavy chain (α-MHC) promoter (pαMHC-EGFP), we investigated the cardiomyogenic effects of 7 synthetic peptides (Betrofin3, FGLs, FGLL, hNgf_C2, EnkaminE, Plannexin and C3) on cardiac differentiation. The expression of several cardiac-specific markers was determined by RT-PCR whereas the structural and functional properties of derived cardiomyocytes were examined by immunofluorescence and electrophysiology, respectively. Results: The results revealed that Betrofin3, an agonist of brain derived neurotrophic factor (BDNF) peptide exerted the most striking pro-cardiomyogenic effect on ES cells. We found that BDNF receptor, TrkB expression was up-regulated during differentiation. Treatment of differentiating cells with Betrofin3 between days 3 and 5 enhanced the expression of cardiac-specific markers and improved cardiomyocyte differentiation and functionality as revealed by genes regulation, flow cytometry and patch clamp analysis. Thus Betrofin3 may exert its cardiomyogenic effects on ES cells via TrkB receptor. Conclusion: Taken together, the results suggest that Betrofin3 modulates BDNF signaling with positive cardiomyogenic effect in stage and dose-dependent manner providing an effective strategy to increase ES cell-based generation of cardiomyocytes and offer a novel therapeutic approach to cardiac pathologies where BDNF levels are impaired.

scholarly journals Nuclear Receptors in Regulation of Mouse ES Cell Pluripotency and Differentiation

PPAR Research ◽  
2007 ◽  
Vol 2007 ◽  
pp. 1-10 ◽  
Author(s):  
Eimear M. Mullen ◽  
Peili Gu ◽  
Austin J. Cooney
Keyword(s):  
Nuclear Receptors ◽  
Therapeutic Potential ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Cell Phenotype ◽  
Es Cell ◽  
Complex Signaling ◽  
Different Cell Types ◽  
Mouse Es Cell

Embryonic stem (ES) cells have great therapeutic potential because they are capable of indefinite self-renewal and have the potential to differentiate into over 200 different cell types that compose the human body. The switch from the pluripotent phenotype to a differentiated cell involves many complex signaling pathways including those involving LIF/Stat3 and the transcription factors Sox2, Nanog and Oct-4. Many nuclear receptors play an important role in the maintenance of pluripotence (ERRβ, SF-1, LRH-1, DAX-1) repression of the ES cell phenotype (RAR, RXR, GCNF) and also the differentiation of ES cells (PPARγ). Here we review the roles of the nuclear receptors involved in regulating these important processes in ES cells.

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