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 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.

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 Fbx15 Is a Novel Target of Oct3/4 but Is Dispensable for Embryonic Stem Cell Self-Renewal and Mouse Development

2003 ◽  
Vol 23 (8) ◽  
pp. 2699-2708 ◽  
Author(s):  
Yoshimi Tokuzawa ◽  
Eiko Kaiho ◽  
Masayoshi Maruyama ◽  
Kazutoshi Takahashi ◽  
Kaoru Mitsui ◽  
...  
Keyword(s):  
Transcription Initiation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Binding Motif ◽  
Es Cell ◽  
Mouse Development ◽  
Specific Expression ◽  
Self Renewal ◽  
Enhancer Element ◽  
Novel Target

ABSTRACT Embryonic stem (ES) cells are immortal and pluripotent cells derived from early mammalian embryos. Transcription factor Oct3/4 is essential for self-renewal of ES cells and early mouse development. However, only a few Oct3/4 target genes have been identified. In this study, we found that F-box-containing protein Fbx15 was expressed predominantly in mouse undifferentiated ES cells. Inactivation of Oct3/4 in ES cells led to rapid extinction of Fbx15 expression. Reporter gene analyses demonstrated that this ES cell-specific expression required an 18-bp enhancer element located approximately 500 nucleotides upstream from the transcription initiation site. The enhancer contained an octamer-like motif and an adjacent Sox-binding motif. Deletion or point mutation of either motif abolished the enhancer activity. The 18-bp fragment became active in NIH 3T3 cells when Oct3/4 and Sox2 were coexpressed. A gel mobility shift assay demonstrated cooperative binding of Oct3/4 and Sox2 to the enhancer sequence. In mice having a β-galactosidase gene knocked into the Fbx15 locus, 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside staining was detected in ES cells, early embryos (two-cell to blastocyst stages), and testis tissue. Despite such specific expression of Fbx15, homozygous mutant mice showed no gross developmental defects and were fertile. Fbx15-null ES cells were normal in morphology, proliferation, and differentiation. These data demonstrate that Fbx15 is a novel target of Oct3/4 but is dispensable for ES cell self-renewal, development, and fertility.

scholarly journals Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

2018 ◽  
Vol 20 (1) ◽  
pp. 19 ◽  
Author(s):  
Yadong Wei ◽  
Krishan Chhiba ◽  
Fengrui Zhang ◽  
Xujun Ye ◽  
Lihui Wang ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mast Cell ◽  
Mast Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Surface Protein ◽  
Cell Types ◽  
Mouse Strains ◽  
Specific Expression

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

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.

182. NOVEL SIGNALLING IN MOUSE EMBRYONIC STEM CELLS GENERATES PRIMITIVE ECTODERM-LIKE CELLS

2009 ◽  
Vol 21 (9) ◽  
pp. 100
Author(s):  
M. B. Morris ◽  
N. Hamra ◽  
A. C. Lonic ◽  
F. Felquer
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Leukaemia Inhibitory Factor ◽  
Signalling Pathways ◽  
Specific Cell ◽  
Self Renewal ◽  
Mouse Es Cells ◽  
Homogeneous Production ◽  
Extracellular Molecules

The phenotypic status of embryonic stem (ES) cells is controlled in part by signalling pathways which translate inputs mediated by extracellular molecules. An important extracellular protagonist in mouse ES cells is LIF (leukaemia inhibitory factor) which interacts with the gp130–LIFR receptor complex to activate a number of downstream signalling pathways, including the STAT3, MEK/ERK and PI3K/Akt. These pathways, together with others, interact in complex and sometimes competing ways to generate the well-known characteristics of mouse ES cells of self-renewal, high rates of proliferation, and pluripotence. The addition of a second molecule, L-proline, to the extracellular environment alters the pluripotent status of mouse ES cells, converting them to a second pluripotent population equivalent to the primitive ectoderm of the pre-gastrulating embryo. This conversion, from ES cells to primitive ectoderm-like cells, primes the latter for directed differentiation to specific cell types (1). Here we show, using inhibitor studies and kinome array analysis, that this small molecule appears to work by (i) changing the balance in activity of signalling pathways already stimulated by LIF and (ii) activating additional signalling pathways. Specifically, L-proline rapidly further activates the LIF-stimulated MEK/ERK pathway, tipping the balance in favour of primitive-ectoderm formation and away from ES-cell self-renewal sustained by LIF-mediated activation of the STAT3 pathway. In addition, L-proline rapidly stimulates other pathways including p38, mTOR and PI3K/Akt each of which contributes, to a greater or lesser extent, to the conversion to primitive ectoderm-like cells. These results indicate that (i) L-proline acts in novel ways to stimulate embryo-like developmental progression in ES cells and (ii) through the addition of small, nontoxic activators and inhibitors of signalling pathways, the differentiation of pluripotent ES cells might be controlled sufficiently well for the homogeneous production of specific cell types suitable for use in animal models of human disease.

Tyrosine kinase signalling in embryonic stem cells

2008 ◽  
Vol 115 (2) ◽  
pp. 43-55 ◽  
Author(s):  
Cecilia Annerén
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Self Renewal ◽  
Mouse Es Cells ◽  
Wide Range

Pluripotent ES (embryonic stem) cells can be expanded in culture and induced to differentiate into a wide range of cell types. Self-renewal of ES cells involves proliferation with concomitant suppression of differentiation. Some critical and conserved pathways regulating self-renewal in both human and mouse ES cells have been identified, but there is also evidence suggesting significant species differences. Cytoplasmic and receptor tyrosine kinases play important roles in proliferation, survival, self-renewal and differentiation in stem, progenitor and adult cells. The present review focuses on the role of tyrosine kinase signalling for maintenance of the undifferentiated state, proliferation, survival and early differentiation of ES cells.

scholarly journals The Polycomb Group Protein Suz12 Is Required for Embryonic Stem Cell Differentiation

2007 ◽  
Vol 27 (10) ◽  
pp. 3769-3779 ◽  
Author(s):  
Diego Pasini ◽  
Adrian P. Bracken ◽  
Jacob B. Hansen ◽  
Manuela Capillo ◽  
Kristian Helin
Keyword(s):  
Cell Differentiation ◽  
Transcriptional Activation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Polycomb Group ◽  
Embryonic Stem Cell Differentiation ◽  
Es Cell ◽  
Polycomb Group Protein ◽  
Specific Expression

ABSTRACT Polycomb group (PcG) proteins form multiprotein complexes, called Polycomb repressive complexes (PRCs). PRC2 contains the PcG proteins EZH2, SUZ12, and EED and represses transcription through methylation of lysine (K) 27 of histone H3 (H3). Suz12 is essential for PRC2 activity and its inactivation results in early lethality of mouse embryos. Here, we demonstrate that Suz12 −/− mouse embryonic stem (ES) cells can be established and expanded in tissue culture. The Suz12 −/− ES cells are characterized by global loss of H3K27 trimethylation (H3K27me3) and higher expression levels of differentiation-specific genes. Moreover, Suz12 −/− ES cells are impaired in proper differentiation, resulting in a lack of repression of ES cell markers as well as activation of differentiation-specific genes. Finally, we demonstrate that the PcGs are actively recruited to several genes during ES cell differentiation, which despite an increase in H3K27me3 levels is not always sufficient to prevent transcriptional activation. In summary, we demonstrate that Suz12 is required for the establishment of specific expression programs required for ES cell differentiation. Furthermore, we provide evidence that PcGs have different mechanisms to regulate transcription during cellular differentiation.

scholarly journals Human embryonic stem cells for brain repair?

2007 ◽  
Vol 363 (1489) ◽  
pp. 87-99 ◽  
Author(s):  
Su-Chun Zhang ◽  
Xue-Jun Li ◽  
M Austin Johnson ◽  
Matthew T Pankratz
Keyword(s):  
Stem Cells ◽  
Motor Neurons ◽  
Es Cells ◽  
Embryonic Stem ◽  
Dopamine Neurons ◽  
Neural Cells ◽  
Es Cell ◽  
Human Es Cells

Cell therapy has been perceived as the main or ultimate goal of human embryonic stem (ES) cell research. Where are we now and how are we going to get there? There has been rapid success in devising in vitro protocols for differentiating human ES cells to neuroepithelial cells. Progress has also been made to guide these neural precursors further to more specialized neural cells such as spinal motor neurons and dopamine-producing neurons. However, some of the in vitro produced neuronal types such as dopamine neurons do not possess all the phenotypes of their in vivo counterparts, which may contribute to the limited success of these cells in repairing injured or diseased brain and spinal cord in animal models. Hence, efficient generation of neural subtypes with correct phenotypes remains a challenge, although major hurdles still lie ahead in applying the human ES cell-derived neural cells clinically. We propose that careful studies on neural differentiation from human ES cells may provide more immediate answers to clinically relevant problems, such as drug discovery, mechanisms of disease and stimulation of endogenous stem cells.

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