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.

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 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.

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 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.

220 NONHUMAN PRIMATE EMBRYONIC STEM CELLS SIMILAR TO THE BIOLOGICAL PROPERTIES OF MOUSE EMBRYONIC STEM CELLS

2012 ◽  
Vol 24 (1) ◽  
pp. 222
Author(s):  
A. Kusanagi ◽  
J. Yamasaki ◽  
C. Iwatani ◽  
H. Tsuchiya ◽  
R. Torii
Keyword(s):  
Nonhuman Primate ◽  
Cynomolgus Monkey ◽  
Es Cells ◽  
Embryonic Stem ◽  
Biological Properties ◽  
Es Cell ◽  
Mouse Es Cells ◽  
Mouse Es Cell

Human and mouse embryonic stem (ES) cells are derived from the inner cell mass of preimplantation blastocysts and human ES cells were long thought to be equivalent to mouse ES cells, despite clear morphological difference and different signalling pathways to maintain their pluripotency between these two ES cell types. Mouse ES cells depend on leukemia inhibitory factor (LIF) and bone morphogenic protein 4 (BMP4) signalling, whereas their human counterparts rely on basic fibroblast growth factor (bFGF) and activin A signalling. The biggest difference of two ES cells is the ability of chimera formation and mouse ES cells can contribute chimera but primate ES cells fails to do that. Monkey ES cells in primates only can be tested for chimera formation in vivo due to the ethical issue and cynomolgus monkey is the most common nonhuman primate to be used for the safety study of drug discoveries. The objective of this study was to develop novel cynomolgus monkey ES cells that have similar biological properties with mouse ES cell and our ultimate goal is to establish germline competent nonhuman primate ES cells. Ovarian stimulation and oocyte collection were carried out for the derivation of ES cells as previously described by Torii et al. Briefly, GnRH (0.9 mg/head) was administered to cynomolgus monkey and two weeks later, a micro infusion pump (iPRECIO™, Primetech Corp) contains FSH was implanted subcutaneously. Follicular aspiration was then performed 40 h after hCG injection and metaphase II oocytes were fertilized by intracytoplasmic sperm injection (ICSI). Cynomolgus monkey ES cells were then established under mouse ES cell conditions such as LIF/STAT signalling and a dome tree-dimensional (3D) morphology nonhuman primate ES cells were selected. On the other hands, ES cells that were established with the presence of basic FGF showed conventional layer-type morphology. Dome-type ES cells express pluripotent transcriptional factors such as Oct-3/4, Nonog and Sox2 as same as layer-type ES cells and both ES lines were capable of multilineage differentiations in vitro after embryoid body formation. Dome-type nonhuman ES cells can also form teratomas and differentiated into all three germ layers when grafted into immunodeficiency mice. For fluorescent gene delivery to nonhuman primate ES cells, feeder-free condition was applied and CAG-GFP vector was transfected into ES cells using Neon electroporation system (Invitrogen Inc.) for the tracing ES cells in the transplantation study. In this study, we have established dome-type ES cell lines that similar to mouse ES cells in morphology and signalling pathway. Dome-type nonhuman primate ES cells express pluripotent gene markers and prove their pluripotency both of in vitro and in vivo, in addition, these modifications would be important to create germline competent ES cells.

scholarly journals UTF1 is a chromatin-associated protein involved in ES cell differentiation

2007 ◽  
Vol 178 (6) ◽  
pp. 913-924 ◽  
Author(s):  
Vincent van den Boom ◽  
Susanne M. Kooistra ◽  
Marije Boesjes ◽  
Bart Geverts ◽  
Adriaan B. Houtsmuller ◽  
...  
Keyword(s):  
Cell Differentiation ◽  
Leucine Zipper ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryonic Cell ◽  
Es Cell ◽  
Self Renewal ◽  
Adult Cell ◽  
Core Histones

Embryonic stem (ES) cells are able to grow indefinitely (self-renewal) and have the potential to differentiate into all adult cell types (pluripotency). The regulatory network that controls pluripotency is well characterized, whereas the molecular basis for the transition from self-renewal to the differentiation of ES cells is much less understood, although dynamic epigenetic gene silencing and chromatin compaction are clearly implicated. In this study, we report that UTF1 (undifferentiated embryonic cell transcription factor 1) is involved in ES cell differentiation. Knockdown of UTF1 in ES and carcinoma cells resulted in a substantial delay or block in differentiation. Further analysis using fluorescence recovery after photobleaching assays, subnuclear fractionations, and reporter assays revealed that UTF1 is a stably chromatin-associated transcriptional repressor protein with a dynamic behavior similar to core histones. An N-terminal Myb/SANT domain and a C-terminal domain containing a putative leucine zipper are required for these properties of UTF1. These data demonstrate that UTF1 is a strongly chromatin-associated protein involved in the initiation of ES cell differentiation.

scholarly journals An Inducible Expression System of the Calcium-Activated Potassium Channel 4 to Study the Differential Impact on Embryonic Stem Cells

2011 ◽  
Vol 2011 ◽  
pp. 1-12 ◽  
Author(s):  
Stefan Liebau ◽  
Michael Tischendorf ◽  
Daniel Ansorge ◽  
Leonhard Linta ◽  
Marianne Stockmann ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cell Differentiation ◽  
Cell Line ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Pacemaker Cell ◽  
Es Cell ◽  
Sk Channel ◽  
Mouse Es Cell

Rationale. The family of calcium-activated potassium channels consists of four members with varying biological functions and conductances. Besides membrane potential modulation, SK channels have been found to be involved in cardiac pacemaker cell development from ES cells and morphological shaping of neural stem cells.Objective. Distinct SK channel subtype expression in ES cells might elucidate their precise impact during cardiac development. We chose SK channel subtype 4 as a potential candidate influencing embryonic stem cell differentiation.Methods. We generated a doxycycline inducible mouse ES cell line via targeted homologous recombination of a cassette expressing a bicistronic construct encoding SK4 and a fluorophore from the murine HPRT locus.Conclusion. We characterized the mouse ES cell line iSK4-AcGFP. The cassette is readily expressed under the control of doxycycline, and the overexpression of SK4 led to an increase in cardiac and pacemaker cell differentiation thereby serving as a unique tool to characterize the cell biological variances due to specific SK channel overexpression.

scholarly journals Human telomeres replicate using chromosome-specific, rather than universal, replication programs

2012 ◽  
Vol 197 (2) ◽  
pp. 253-266 ◽  
Author(s):  
William C. Drosopoulos ◽  
Settapong T. Kosiyatrakul ◽  
Zi Yan ◽  
Simone G. Calderano ◽  
Carl L. Schildkraut
Keyword(s):  
Single Molecule ◽  
Embryonic Stem ◽  
Cell Types ◽  
Es Cell ◽  
Site Location ◽  
Subtelomeric Heterochromatin ◽  
Basic Features ◽  
Different Cell Types ◽  
Single Molecule Analysis ◽  
Individual Human

Telomeric and adjacent subtelomeric heterochromatin pose significant challenges to the DNA replication machinery. Little is known about how replication progresses through these regions in human cells. Using single molecule analysis of replicated DNA (SMARD), we delineate the replication programs—i.e., origin distribution, termination site location, and fork rate and direction—of specific telomeres/subtelomeres of individual human chromosomes in two embryonic stem (ES) cell lines and two primary somatic cell types. We observe that replication can initiate within human telomere repeats but was most frequently accomplished by replisomes originating in the subtelomere. No major delay or pausing in fork progression was detected that might lead to telomere/subtelomere fragility. In addition, telomeres from different chromosomes from the same cell type displayed chromosome-specific replication programs rather than a universal program. Importantly, although there was some variation in the replication program of the same telomere in different cell types, the basic features of the program of a specific chromosome end appear to be conserved.

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.

Sign in / Sign up

Export Citation Format

Share Document