scholarly journals C3G Regulates STAT3, ERK, Adhesion Signaling, and Is Essential for Differentiation of Embryonic Stem Cells

2021 ◽  
Author(s):  
Vijay V. Vishnu ◽  
Bh. Muralikrishna ◽  
Archana Verma ◽  
Sanjeev Chavan Nayak ◽  
Divya Tej Sowpati ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Erk Activity

SummaryC3G (RAPGEF1), engaged in multiple signaling pathways, is essential for the early development of the mouse. In this study, we have examined its role in mouse embryonic stem cell self-renewal and differentiation. C3G null cells generated by CRISPR mediated knock-in of a targeting vector exhibited enhanced clonogenicity and long-term self-renewal. They did not differentiate in response to LIF withdrawal when compared to the wild type ES cells and were defective for lineage commitment upon teratoma formation in vivo. Gene expression analysis of C3G KO cells showed misregulated expression of a large number of genes compared with WT cells. They express higher levels of self-renewal factors like KLF4 and ESRRB and show high STAT3 activity, and very low ERK activity compared to WT cells. Reintroduction of C3G expression in a KO line partially reverted expression of ESRRB, and KLF4, and ERK activity similar to that seen in WT cells. The expression of self-renewal factors was persistent for a longer time, and induction of lineage-specific markers was not seen when C3G KO cells were induced to form embryoid bodies. C3G KO cells showed poor adhesion and significantly reduced levels of pFAK, pPaxillin, and Integrin-β1, in addition to downregulation of the cluster of genes involved in cell adhesion, compared to WT cells. Our results show that C3G is essential for the regulation of STAT3, ERK, and adhesion signaling, to maintain pluripotency of mouse embryonic stem cells and enable their lineage commitment for differentiation. Graphical abstract

scholarly journals Self-Renewal Versus Lineage Commitment of Embryonic Stem Cells: Protein Kinase C Signaling Shifts the Balance

Stem Cells ◽  
2011 ◽  
Vol 29 (4) ◽  
pp. 618-628 ◽  
Author(s):  
Debasree Dutta ◽  
Soma Ray ◽  
Pratik Home ◽  
Melissa Larson ◽  
Michael W. Wolfe ◽  
...  
Keyword(s):  
Stem Cells ◽  
Protein Kinase C ◽  
Embryonic Stem Cells ◽  
Protein Kinase ◽  
Embryonic Stem ◽  
Lineage Commitment ◽  
Self Renewal ◽  
Kinase C

230 GENERATION OF PUTATIVE RABBIT EMBRYONIC STEM CELLS

2007 ◽  
Vol 19 (1) ◽  
pp. 231
Author(s):  
S. Wang ◽  
X. Tang ◽  
Y. Niu ◽  
H. Chen ◽  
T. Li ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
High Speed ◽  
Es Cells ◽  
Research Work ◽  
Embryonic Stem ◽  
Morphological Characteristics ◽  
Mouse Escs

The rabbit, as a laboratory animal model, has several advantages in the study of human physiological disorders. In this study, stable putative pluripotent rabbit embryonic stem cells (rESCs) were derived from in vivo-fertilized and in vitro-cultured blastocysts. The rabbit ICMs were obtained by 0.05% trypsin–0.008% EDTA treatment and mechanical separation; the ES-like cell colonies seen several days later. ICM-derived outgrowths which were treated with 5 mg/mL-1 dispase, followed by 0.05% trypsin–0.008% EDTA, were mechanically disaggregated into small clumps and reseeded on MEFs. The putative ES cell lines maintained expression of pluripotent cells markers and normal XY karyotype for long periods of culture (>1 month). The putative rESCs expressed alkaline phosphatase, transcription factor Oct-4, stage-specific embryonic antigens (SSEA-1, SSEA-3, and SSEA-4), and tumor-related antigens (TRA-1-60 and TRA-1-81). The morphological characteristics of the putative ESCs are closer to those of human ESCs; their high speed of proliferation, however, is closer to that of mouse ESCs. Putative rabbit ESCs were induced to differentiate into many cell types including trophoblast cells, similar to primate ESCs, in vitro, and formed teratomas with derivatives of the 3 major germ layers in vivo when injected into SCID mice. Using RT-PCR measurement, but with some differences in ligands and inhibitors, and comparing with human and mouse ESCs, the putative rabbit ESCs expressed similar genes related to pluripotency (Oct-4, Nanog, SOX2, and UTF-1) and similar genes of FGF, WNT, and TGF signaling pathways related to the proliferation and self-renewal. Our further research work showed that TGF beta and FGF pathways cooperate to maintain pluripotency of rabbit ESCs similar to those of human ES cells.

scholarly journals RUNX1a enhances hematopoietic lineage commitment from human embryonic stem cells and inducible pluripotent stem cells

Blood ◽  
2013 ◽  
Vol 121 (15) ◽  
pp. 2882-2890 ◽  
Author(s):  
Dan Ran ◽  
Wei-Jong Shia ◽  
Miao-Chia Lo ◽  
Jun-Bao Fan ◽  
David A. Knorr ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Pluripotent Stem Cells ◽  
Ex Vivo ◽  
Ectopic Expression ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Lineage Commitment ◽  
Hematopoietic Differentiation

Abstract Advancements in human pluripotent stem cell (hPSC) research have potential to revolutionize therapeutic transplantation. It has been demonstrated that transcription factors may play key roles in regulating maintenance, expansion, and differentiation of hPSCs. In addition to its regulatory functions in hematopoiesis and blood-related disorders, the transcription factor RUNX1 is also required for the formation of definitive blood stem cells. In this study, we demonstrated that expression of endogenous RUNX1a, an isoform of RUNX1, parallels with lineage commitment and hematopoietic emergence from hPSCs, including both human embryonic stem cells and inducible pluripotent stem cells. In a defined hematopoietic differentiation system, ectopic expression of RUNX1a facilitates emergence of hematopoietic progenitor cells (HPCs) and positively regulates expression of mesoderm and hematopoietic differentiation-related factors, including Brachyury, KDR, SCL, GATA2, and PU.1. HPCs derived from RUNX1a hPSCs show enhanced expansion ability, and the ex vivo–expanded cells are capable of differentiating into multiple lineages. Expression of RUNX1a in embryoid bodies (EBs) promotes definitive hematopoiesis that generates erythrocytes with β-globin production. Moreover, HPCs generated from RUNX1a EBs possess ≥9-week repopulation ability and show multilineage hematopoietic reconstitution in vivo. Together, our results suggest that RUNX1a facilitates the process of producing therapeutic HPCs from hPSCs.

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.

Hematopoietic Development from Human Embryonic Stem Cells

Hematology ◽  
2007 ◽  
Vol 2007 (1) ◽  
pp. 11-16 ◽  
Author(s):  
Mickie Bhatia
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Cell Fate ◽  
Human Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Immunodeficient Mice ◽  
Cell Based Therapy

Abstract The most common human cell-based therapy applied today is hematopoietic stem cell (HSC) transplantation. HSCs can be defined by two essential properties: self-renewal and multilineage hematopoietic differentiation. These combined HSC properties allow them to differentiate into all blood cell types (multilineage) in a sustained manner for the lifetime of the animal, which requires their ability to make cellular copies of themselves (self-renewal). These features can be tested by transplantation from donor to recipient and provide a functional basis to define and identify HSCs. Currently, human bone marrow (BM), mobilized peripheral blood, and umbilical cord blood (CB) represent the major sources of transplantable HSCs, but their availability for use is limited by both quantity and compatibility. Although increasing evidence suggests that somatic HSCs can be expanded to meet current needs, their in vivo potential is concomitantly compromised after ex vivo culture. Pluripotent human embryonic stem cells (hESCs) may provide an alternative. hESCs possess indefinite proliferative capacity in vitro, and have been shown to differentiate into the hematopoietic cell fate, giving rise to erythroid, myeloid, and lymphoid lineages using a variety of differentiation procedures. In most cases, hESC-derived hematopoietic cells show similar clonogenic progenitor capacity and primitive phenotype to somatic sources of hematopoietic progenitors, but possess limited in vivo repopulating capacity when transplanted into immunodeficient mice. Although this suggests HSC function can be derived from hESCs, the efficiency and quality of these cells must be characterized using surrogate models for potential clinical applications.

392 EFFECTS OF TRICHOSTATIN A, AN EPIGENETIC MODIFIER AGENT, ON DIFFERENTIATION OF EMBRYONIC STEM CELLS INTO STRIATED MUSCLE CELLS

2010 ◽  
Vol 22 (1) ◽  
pp. 352
Author(s):  
C. S. Oliveira ◽  
N. Z. Saraiva ◽  
Jasmin ◽  
M. M. Souza ◽  
T. A. D. Tetzner
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Troponin I ◽  
Striated Muscle ◽  
Trichostatin A ◽  
Es Cells ◽  
Embryonic Stem ◽  
Muscle Cells ◽  
Embryoid Bodies ◽  
Significance Level

In vitro generation of cardiomyocytes from embryonic stem cells (ES cells) is a promising approach to develop strategies for treatment of cardiac diseases. Epigenetic changes occur during ES cells differentiation, and by the first 5 days, the histone acetylation levels increase, promoting an improvement in gene expression. Trichostatin A (TSA) is a histone deacetylase (HDAC) inhibitor and promotes histone hyperacetylation. In this study, we analyzed the effects of TSA treatment in ES cells differentiation into striated muscle cells. For that, murine ES cell line H106 was grown in hanging drops of 20 μL containing 2000 cells in DMEM medium supplemented with 15% FCS, 10 mM 2-mercaptoethanol, 1 mM sodium pyruvate, 2 m L-glutamine, 10 mM nonessential amino acids, and 83.4 μg mL-1 amikacin. After 5 days, embryoid bodies were transferred individually to a 96-well plate treated with 0.1% swine gelatin. Trichostatin A treatment was performed during hanging drop culture (group 15 nM d0-5), at Day 5 for 24 h after transfer to adherent culture (groups 50 nM d5 and 100 nM d5), and at Day 13 for 24 h (groups 50 nM d13 and 100 nM d13). Area of embryoid bodies and apoptosis rate from control and 15 nM d0-5 groups were analyzed at Day 5. Analysis of contractile structures was carried out at Day 14. Imunnocitochemistry reactions for desmin and troponin I were performed at Day 7 and 17, respectively. Results of apoptosis, desmin, and troponin I cell rates (positive cells/total cells) were analyzed by chi-square test, with a significance level of 5%, on MINITAB Release 14.1. Areas of embryoid bodies were submitted to one-way ANOVA and Tukey’s post-test, with a significance level of 5%, using GraphPad software. Embryoid bodies developed on TSA supplemented medium presented smaller areas (15 nM d0-5: 6.75 ± 0.93 mm2; control: 15.84 ± 1.64 mm2) and greater apoptosis rates (15 nM d0-5: 29.53%; control: 20.18%). Contractile structures were greater on 50 nM d5 (90%c) and extremely less on the 15 nM d0-5 group (3.12%b). Groups 100 nM d5 (66.6%), 50 nM d13 (70.93%), and 100 nM d13 (80.7%a,c) were similar to the control group (68.25%a). Rate of desmin positive cells was greater on the 50 nM d5 group (31.53b) and less on the 100 nM d5 group (22.9c). The 15 nM d0-5 group (26.03a) was similar to control (25.25a). Rate of troponin I positive cells was greater on 50 nM d5 (8.65b) and 100 nM d13 (9.69b) and less on the 100 nM d5 group (2.63c). On the 15 nM d0-5 group, no positive cells were observed, and the 50 nM d13 group (6.67a) was similar to control (6.44a). In conclusion, the current study demonstrated that TSA improves striated muscle differentiation when supplemented at lesser concentrations at Day 5 (50 nM) and greater concentrations at Day 13 (100 nM) and promotes detrimental effects when used during embryoid body development, decreasing the area of structures and increasing apoptosis rate. Acknowledgments are given to FAPESP 2007/55968-9 and 2008/58370-0.

scholarly journals Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells

2008 ◽  
Vol 205 (8) ◽  
pp. 1917-1927 ◽  
Author(s):  
Hidekazu Nishikii ◽  
Koji Eto ◽  
Noriko Tamura ◽  
Koichi Hattori ◽  
Beate Heissig ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Blood Platelets ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Normal Platelet ◽  
Metalloproteinase Inhibitors ◽  
Flow Cytometric Sorting

Embryonic stem cells (ESCs) could potentially compensate for the lack of blood platelets available for use in transfusions. Here, we describe a new method for generating mouse ESC-derived platelets (ESPs) that can contribute to hemostasis in vivo. Flow cytometric sorting of cells from embryoid bodies on day 6 demonstrated that c-Kit+ integrin αIIb (αIIb)+ cells, but not CD31+ cells or vascular endothelial cadherin+ cells, are capable of megakaryopoiesis and the release of platelet-like structures by day 12. αIIbβ3-expressing ESPs exhibited ectodomain shedding of glycoprotein (GP)Ibα, GPV, and GPVI, but not αIIbβ3 or GPIbβ. ESPs showed impaired αIIbβ3 activation and integrin-mediated actin reorganization, critical events for normal platelet function. However, the administration of metalloproteinase inhibitors GM6001 or TAPI-1 during differentiation increased the expression of GPIbα, improving both thrombogenesis in vitro and posttransfusion recovery in vivo. Thus, the regulation of metalloproteinases in culture could be useful for obtaining high-quality, efficacious ESPs as an alternative platelet source for transfusions.

Thrombopoietin (TPO) Enhances Heamatopoietic Differentiation of Human Embryonic Stem Cells.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4157-4157
Author(s):  
Anand S. Srivastava ◽  
Rangrath Mishra ◽  
Ewa Carrier
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Es Cells ◽  
Embryonic Stem ◽  
Cytoplasmic Domain ◽  
Embryoid Bodies ◽  
Janus Kinase ◽  
Human Es Cells

Abstract Recently, it was demonstrated that TPO enhances hematopoietic differentiation of primate ES cells, but its role in differentiating human ES cells is unknown. We sought to investigate the regulatory mechanism of TPO induced signals mediated by the c-mpl cytoplasmic domain during human embryonic stem (hES) cells hematopoietic commitment. We hypothesize that in human embryonic stem cells, binding of TPO to its c-mpl receptor causes three-dimensional alterations which bring the c-mpl cytoplasmic domain and Janus Kinase into close-proximity and thus induces the phosphorylation and dimerization of STAT5 molecule. Dimerized STAT5 molecules detach from the receptors and migrate to the nucleus where they bind GAS site and induce transcription of a set of target, hematopoiesis-related genes. NIH human ES cell lines (WI01) were used in this experiment. In brief, to induce EB formation, cells were incubated in differentiation medium, which consisted of knockout DMEM medium (GIBCO/BRL, Carlsbad, USA), supplemented with 20% non-heat-inactivated fetal bovine serum (FBS, Hyclone, USA), 1% nonessential amino acids, 1 mM L-glutamine, and 0.1 mM β-mercaptoethanol. Subsequently, DMEM was replaced by IMDM (GIBCO/BRL, USA) with the same supplements and additional two cytokines (100 ng/mL SCF and 100 ng/mL Flt-3 ligand (Flt-3L)) (control group). To investigate the role of TPO and VEGF, cells were additionally treated with 100 ng/mL TPO alone or in combination with 100 ng/mL rhVEGF. All cytokines were from the R&D systems (USA). Significant increase in the numbers of embryoid bodies (EBs) formation in TPO (125/105), TPO/VEGF (150/105 cells) when compared to controls (10/105 planted ES cells) was documented. This corresponded with the increase in CFU-C and the number of CD31/CD34 positive and CD34-positive progenitors. Analysis of gene expression during hematopoietic development demonstrated that TPO/VEGF combination increased mRNA expression of the TPO receptor (TPO-R) and VEGF (VEGF-R) receptors in hematopoietic progenitors obtained from human ES cells. We are in the process of determining the role of JAK/STAT pathway in this process; functional studies involve blocking of TPO/c-mpl using TPO-R-specific antibodies and determining its impact on human ES-derived hematopoiesis.

scholarly journals Comparison of Teratoma Formation between Embryonic Stem Cells and Parthenogenetic Embryonic Stem Cells by Molecular Imaging

2018 ◽  
Vol 2018 ◽  
pp. 1-9 ◽  
Author(s):  
Hongyan Tao ◽  
Xiaoniao Chen ◽  
Anbang Wei ◽  
Xianghe Song ◽  
Weiqiang Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Embryonic Stem ◽  
Growth Factor Receptor ◽  
Renilla Luciferase ◽  
Alternative Source ◽  
Teratoma Formation

With their properties of self-renewal and differentiation, embryonic stem (ES) cells hold great promises for regenerative therapy. However, teratoma formation and ethical concerns of ES cells may restrict their potential clinical applications. Currently, parthenogenetic embryonic stem (pES) cells have attracted the interest of researchers for its self-renewing and pluripotent differentiation while eliciting less ethic concerns. In this study, we established a model with ES and pES cells both stably transfected with a double-fusion reporter gene containing renilla luciferase (Rluc) and red fluorescent protein (RFP) to analyze the mechanisms of teratoma formation. Transgenic Vegfr2-luc mouse, which expresses firefly luciferase (Fluc) under the promoter of vascular endothelial growth factor receptor 2 (Vegfr2-luc), was used to trace the growth of new blood vessel recruited by transplanted cells. Bioluminescence imaging (BLI) of Rluc/Fluc provides an effective tool in estimating the growth and angiogenesis of teratoma in vivo. We found that the tumorigenesis and angiogenesis capacity of ES cells were higher than those of pES cells, in which VEGF/VEGFR2 signal pathway plays an important role. In conclusion, pES cells have the decreased potential of teratoma formation but meanwhile have similar differentiating capacity compared with ES cells. These data demonstrate that pES cells provide an alternative source for ES cells with the risk reduction of teratoma formation and without ethical controversy.

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