Role of the phosphoinositide 3-kinase pathway in mouse embryonic stem (ES) cells

2005 ◽  
Vol 33 (6) ◽  
pp. 1522 ◽  
Author(s):  
M. Murakami ◽  
K. Takahashi ◽  
S. Yamanaka
Keyword(s):  
Es Cells ◽  
Embryonic Stem ◽  
Phosphoinositide 3 Kinase ◽  
Kinase Pathway

Role of the phosphoinositide 3-kinase pathway in mouse embryonic stem (ES) cells

2005 ◽  
Vol 33 (6) ◽  
pp. 1522-1525 ◽  
Author(s):  
K. Takahashi ◽  
M. Murakami ◽  
S. Yamanaka
Keyword(s):  
Es Cells ◽  
Mammalian Target Of Rapamycin ◽  
Embryonic Stem ◽  
Pi3k Pathway ◽  
Leukaemia Inhibitory Factor ◽  
Mouse Es Cells ◽  
Ras Family ◽  
Phosphoinositide 3 Kinase ◽  
Kinase Pathway

Mouse ES (embryonic stem) cells maintain pluripotency with robust proliferation in vitro. ES cells share some similarities with cancer cells, such as anchorage-independent growth, loss of contact inhibition and tumour formation. After differentiation, ES cells lose pluripotency and tumorigenicity. Recent studies showed that the PI3K (phosphoinositide 3-kinase) pathway is important for proliferation, survival and maintenance of pluripotency in ES cells. The PI3K pathway is activated by growth factors and cytokines including insulin and leukaemia inhibitory factor. In addition to these exogenous factors, the PI3K pathway is endogenously activated by the constitutively active Ras family protein ERas (ES cell-expressed Ras). The PI3K pathway utilizes multiple downstream effectors including mTOR (mammalian target of rapamycin), which we have shown to be essential for proliferation in mouse ES cells and early embryos.

Abstract 361: Creg1 Interacts With Sec8 to Promote Cell-cell Adhesion During Cardiomyogenesis

2016 ◽  
Vol 119 (suppl_1) ◽  
Author(s):  
Jie Liu ◽  
Yanmei Qi ◽  
Shu-Chan Hsu ◽  
Siavash Saadat ◽  
Saum Rahimi ◽  
...  
Keyword(s):  
Cell Adhesion ◽  
Es Cells ◽  
Embryonic Stem ◽  
Intercellular Junctions ◽  
Amino Acid Residues ◽  
Loss Of Function ◽  
Wild Type ◽  
Adhesion Sites ◽  
Cell Cell

Cellular repressor of E1A-stimulated genes 1 (CREG1) is a 24 kD glycoprotein essential for early embryonic development. Our immunofluorescence studies revealed that CREG1 is highly expressed at myocyte junctions in both embryonic and adult hearts. To explore it role in cardiomyogenesis, we employed gain- and loss-of-function analyses demonstrating that CREG1 is required for the differentiation of mouse embryonic stem (ES) cell into cohesive myocardium-like structures. Chimeric cultures of wild-type and CREG1 knockout ES cells expressing cardiac-specific reporters showed that the cardiomyogenic effect of CREG1 is cell autonomous. Furthermore, we identified a novel interaction between CREG1 and Sec8 of the exocyst complex, which tethers vesicles to the plasma membrane. Mutations of the amino acid residues D141 and P142 to alanine in CREG1 abolished its binding to Sec8. To address the role of the CREG1-Sec8 interaction in cardiomyogenesis, we rescued CREG1 knockout ES cells with wild-type and Sec8-binding mutant CREG1 and showed that CREG1 binding to Sec8 promotes cardiomyocyte differentiation and cohesion. Mechanistically, CREG1, Sec8 and N-cadherin all localize at cell-cell adhesion sites. CREG1 overexpression enhances the assembly of adherens and gap junctions. By contrast, its knockout inhibits the Sec8-N-cadherin interaction and induces their degradation. Finally, shRNA-mediated knockdown of Sec8 leads to cardiomyogenic defects similar to CREG1 knockout. These results suggest that the CREG1 binding to Sec8 enhances the assembly of intercellular junctions and promotes cardiomyogenesis.

scholarly journals Inflammation increases cells expressing ZSCAN4 and progenitor cell markers in the adult pancreas

2013 ◽  
Vol 304 (12) ◽  
pp. G1103-G1116 ◽  
Author(s):  
Shigeru B. H. Ko ◽  
Sakiko Azuma ◽  
Yukihiro Yokoyama ◽  
Akiko Yamamoto ◽  
Kazuhiro Kyokane ◽  
...  
Keyword(s):  
Autoimmune Pancreatitis ◽  
Genome Stability ◽  
Progenitor Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
Corticosteroid Treatment ◽  
Human Pancreas ◽  
Cell Markers ◽  
Telomere Elongation

We have recently identified the zinc finger and SCAN domain containing 4 (Zscan4), which is transiently expressed and regulates telomere elongation and genome stability in mouse embryonic stem (ES) cells. The aim of this study was to examine the expression of ZSCAN4 in the adult pancreas and elucidate the role of ZSCAN4 in tissue inflammation and subsequent regeneration. The expression of ZSCAN4 and other progenitor or differentiated cell markers in the human pancreas was immunohistochemically examined. Pancreas sections of alcoholic or autoimmune pancreatitis patients before and under maintenance corticosteroid treatment were used in this study. In the adult human pancreas a small number of ZSCAN4-positive (ZSCAN4+) cells are present among cells located in the islets of Langerhans, acini, ducts, and oval-shaped cells. These cells not only express differentiated cell markers for each compartment of the pancreas but also express other tissue stem/progenitor cell markers. Furthermore, the number of ZSCAN4+cells dramatically increased in patients with chronic pancreatitis, especially in the pancreatic tissues of autoimmune pancreatitis actively regenerating under corticosteroid treatment. Interestingly, a number of ZSCAN4+cells in the pancreas of autoimmune pancreatitis returned to the basal level after 1 yr of maintenance corticosteroid treatment. In conclusion, coexpression of progenitor cell markers and differentiated cell markers with ZSCAN4 in each compartment of the pancreas may indicate the presence of facultative progenitors for both exocrine and endocrine cells in the adult pancreas.

scholarly journals Developmental regulation of yolk sac hematopoiesis by Krüppel-like factor 6

Blood ◽  
2006 ◽  
Vol 107 (4) ◽  
pp. 1357-1365 ◽  
Author(s):  
Nobuyuki Matsumoto ◽  
Atsushi Kubo ◽  
Huixian Liu ◽  
Kuniharu Akita ◽  
Friedrich Laub ◽  
...  
Keyword(s):  
Developmental Regulation ◽  
Es Cells ◽  
Embryonic Stem ◽  
Early Embryo ◽  
Yolk Sac ◽  
Homozygous Mutation ◽  
Mesoderm Induction ◽  
Mrna Levels ◽  
Mouse Development

Krüppel-like factor 6 (KLF6) is a member of a growing family of transcription factors that share a common 3 C2H2 zinc finger DNA binding domain and have broad activity in regulating proliferation and development. We have previously established that Klf6 is expressed in neuronal tissue, hindgut, heart, lung, kidney, and limb buds during midgestation. To explore the potential role of Klf6 in mouse development, we analyzed Klf6-/- mice and found that the homozygous mutation is embryonic lethal by embryonic day (E) 12.5 and associated with markedly reduced hematopoiesis and poorly organized yolk sac vascularization. Additionally, mRNA levels of Scl and Gata1 were reduced by approximately 80% in Klf6-/- yolk sacs. To further analyze this phenotype, we generated Klf6-/- embryonic stem (ES) cells by homologous recombination, and compared their capacity to differentiate into the hematopoietic lineage with that of either Klf6+/- or Klf6+/+ ES cells. Consistent with the phenotype in the early embryo, Klf6-/- ES cells displayed significant hematopoietic defects following differentiation into EBs. Prolongation of epiblast-like cells and delays in mesoderm induction were also observed in the Klf6-/- EBs, associated with delayed expression of Brachyury, Klf1, and Gata1. Forced expression of KLF6 using a tet-inducible system enhanced the hematopoietic potential of wild-type EBs. Collectively, these findings implicate Klf6 in ES-cell differentiation and hematopoiesis.

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.

Analysis of the role of interleukin-1β (IL-1β)in vivo by gene targeting in embryonic stem (ES) cells

Cytokine ◽  
1994 ◽  
Vol 6 (5) ◽  
pp. 574
Author(s):  
L. Shornick ◽  
P. De Togni ◽  
S. Mariathasan ◽  
A. Fick ◽  
J. Goellner ◽  
...  
Keyword(s):  
Gene Targeting ◽  
Es Cells ◽  
Embryonic Stem ◽  
Interleukin 1Β

scholarly journals MOZ and BMI1 play opposing roles during Hox gene activation in ES cells and in body segment identity specification in vivo

2015 ◽  
Vol 112 (17) ◽  
pp. 5437-5442 ◽  
Author(s):  
Bilal N. Sheikh ◽  
Natalie L. Downer ◽  
Belinda Phipson ◽  
Hannah K. Vanyai ◽  
Andrew J. Kueh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Gene Activation ◽  
Embryonic Stem ◽  
Mrna Levels ◽  
Body Segment ◽  
Initial Activation ◽  
Activation Phase

Hox genes underlie the specification of body segment identity in the anterior–posterior axis. They are activated during gastrulation and undergo a dynamic shift from a transcriptionally repressed to an active chromatin state in a sequence that reflects their chromosomal location. Nevertheless, the precise role of chromatin modifying complexes during the initial activation phase remains unclear. In the current study, we examined the role of chromatin regulators during Hox gene activation. Using embryonic stem cell lines lacking the transcriptional activator MOZ and the polycomb-family repressor BMI1, we showed that MOZ and BMI1, respectively, promoted and repressed Hox genes during the shift from the transcriptionally repressed to the active state. Strikingly however, MOZ but not BMI1 was required to regulate Hox mRNA levels after the initial activation phase. To determine the interaction of MOZ and BMI1 in vivo, we interrogated their role in regulating Hox genes and body segment identity using Moz;Bmi1 double deficient mice. We found that the homeotic transformations and shifts in Hox gene expression boundaries observed in single Moz and Bmi1 mutant mice were rescued to a wild type identity in Moz;Bmi1 double knockout animals. Together, our findings establish that MOZ and BMI1 play opposing roles during the onset of Hox gene expression in the ES cell model and during body segment identity specification in vivo. We propose that chromatin-modifying complexes have a previously unappreciated role during the initiation phase of Hox gene expression, which is critical for the correct specification of body segment identity.

scholarly journals Fetal and trophoblast PI3Kp110α have distinct roles in regulating resource supply to the growing fetus

2018 ◽  
Author(s):  
Jorge Lopez-Tello ◽  
Vicente Perez-Garcia ◽  
Jaspreet Khaira ◽  
Laura C. Kusinski ◽  
Wendy N. Cooper ◽  
...  
Keyword(s):  
Fetal Growth ◽  
Embryonic Stem ◽  
Prenatal Development ◽  
Complete Loss ◽  
Placental Development ◽  
Expression Of Genes ◽  
Transport Loss ◽  
Healthy Growth ◽  
Phosphoinositide 3 Kinase

AbstractPrevious studies suggest that the placental supply of nutrients to the fetus adapts according to fetal demand. However, the signaling events underlying placental adaptations remain largely unknown. Earlier work in mice has revealed that loss of the phosphoinositide 3-kinase p110α impairs feto-placental growth but placental nutrient supply is adaptively increased. Here we explore the role of p110α in the epiblast-derived (fetal) and trophoblast lineages of the conceptus in relation to feto-placental growth and placental development and transfer function. Using conditional gene manipulations to knock-down p110α either by ∼50% or ∼100% in the fetal lineages and/or trophoblast, this study shows that p110α in the fetus is essential for prenatal development and a major regulator of placental phenotype in mice. Complete loss of fetal p110α caused embryonic death, whilst heterozygous loss resulted in fetal growth restriction and impaired placental formation and nutrient transport. Loss of trophoblast p110α also resulted in abnormal placental development, although fetuses were viable. However, in response to complete loss of trophoblast p110α, the placenta failed to transport sufficient amino acid to match fetal demands for growth. Using RNA-seq, we identified several genes downstream of p110α in the trophoblast that are important in adapting placental phenotype to support fetal growth. Further work using CRISPR/Cas9 genome targeting showed that loss of p110α differentially affects the expression of genes in trophoblast and embryonic stem cells. Our findings thus reveal important, but distinct roles for p110α signaling in the different compartments of the conceptus, which control fetal resource acquisition and ultimately affect healthy growth.One Sentence SummaryFetal and trophoblast p110α modify resource allocation

Role of TPO in the Differentiation of Embryonic Stem Cells into Hematopoetic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4202-4202
Author(s):  
Zheng Wang ◽  
Pramono Andri ◽  
Skokowa Julia ◽  
Welte Karl
Keyword(s):  
Stem Cell ◽  
Cell Differentiation ◽  
Rhesus Monkey ◽  
Embryonic Stem Cell ◽  
Mrna Expression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryonic Stem Cell Differentiation

Abstract Thrombopoetin (TPO) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice and in congenital amegakaryocytic thrombocytopenia-patients with non-sense c-mpl mutation who develop pancytopenia during the first years of life suggest that TPO also play an important role on early hematopoesis. We demonstrated that TPO enhances FLK-1 (VEGF-receptor) expression on hemangioblasts during murine embryonic stem cell differentiation in embryoid body-liquid cultures (up to 73%). To extend our studies, we investigated the TPO signaling in FLK-1 positive cells. ES cells at different time point of differentiation showed that TPO enhances c-mpl-, BMP4-, Notch-, HOXB4-, HOXB9-, HOXA10-, Runx1-and CD133- mRNA expression. To investigate mesoderm formation, we also analyzed GATA-4 and T-brachyury mRNA level expression. Interestingly, we found that TPO alone did not increase GATA-4- and T-brachyury- mRNA expression, suggesting that TPO requires other cytokines to form the mesoderm. We also found that TPO could maintain VEGF-A mRNA expression level during differentiation of ES-cells. We hypothesize that VEGF expression together with c-mpl expression is required in hematopoetic differentiation of ES cell. This activity of Tpo was also observed during Rhesus monkey embryonic stem cell differentiation into hematopoetic cell. Only combinations of TPO and VEGF were capable of increasing CD34 positive hematopoietic progenitor cells (up to 8%), but TPO alone failed to induce high levels of CD34+ cell. In addition, analysis of gene expression during hemangioblast development demonstrated that TPO was capable of increasing the expression of VEGF receptors (FLK-1) and TPO receptors (c-mpl) in mice and primates. The in-vitro differentiation of mouse and rhesus monkey ES cells provides an opportunity to better understand the role of TPO in the early stage of hematopoietic development from ES cells to mature hematopoietic cells.

Filamins Regulate Differentiation of Megakaryocytes and Platelets From Cultured Embryonic Stem (ES) Cells.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1618-1618 ◽  
Author(s):  
Taisuke Kanaji ◽  
Takashi Okamura ◽  
Peter J. Newman
Keyword(s):  
Cell Differentiation ◽  
Board Of Directors ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Actin Binding ◽  
Wild Type ◽  
Advisory Committees ◽  
Hematopoietic Stem

Abstract Abstract 1618 Filamins (Flns) A and B are major non-muscle actin binding proteins that play important roles in cross-linking cortical actin filaments into three-dimensional networks. In addition to their role as cytoskeletal scaffolding molecules, filamins are also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. The role of filamins in hematopoietic stem cell differentiation, however, remains unclear, in part because gene-targeted mice lacking filamins die early on in embryonic development. To investigate the role of filamins A and B in the differentiation of embryonic stem cells (ESCs) along the megakaryocyte/platelet axis, we designed shRNA-containing vectors that targeted both FlnA and B under the control of either the CMV immediate-early promoter (CMV-FlnABLow), or an endogenous Rosa26 promoter (Rosa26-FlnABLow). Compared with wild-type ESCs, FlnABLow ESCs formed small, tightly packed undifferentiated colonies that expressed high levels of the ESC transcription factor, Nanog, and low levels of ERK activity – all indicators of an undifferentiated state. Embryoid prepared from FlnABLow ESCs, were allowed to differentiate, and examined for markers of mesoderm differentiation (Flk-1) and megakaryocyte differentiation (CD41). Whereas Day 6 EB-derived FlnABnormal wild-type cells were 8% Flk-1 positive and 13% CD41 positive, Day 6 FlnABLow cells were 9% Flk-1 positive and only 4% CD41 positive, consistent with the notion that loss of Fln A and B results in a delay of mesoderm to hematopoietic differentiation. To evaluate the effect of Fln knockdown on the ability of the CD41-positive cells to further differentiate into megakaryocytes, form proplatelet extensions, and produce platelets, CD41 positive cells isolated from day 8 EBs were cultured in the presence of a thrombopoietin (TPO)-producing TERT stromal cell line. We found that FlnABLow CD41-positive cells formed far fewer and smaller megakaryocytes compared with their FlnABnormal wild-type counterparts. Proplatelets derived from FlnABLow cells exhibited an abnormal, enlarged morphology with swellings and thick shafts that released platelets prematurely, yielding platelets that were nearly twice the size of those derived from FlnABnormal cells. Taken together, we conclude that not only do filamins function prominently in hematopoietic cell differentiation, they also play an important role in platelet production, likely via their ability to by recruit and organize the necessary signaling molecules near the inner face of the plasma membrane. Disclosures: Newman: New York Blood Center: Membership on an entity's Board of Directors or advisory committees; Children's Hospital of Boston: Membership on an entity's Board of Directors or advisory committees.

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