Abstract 441: Role of Nitric Oxide and S-nitrosylation in Cardiomyogenesis by iPSCs

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Alessandro Salerno ◽  
Konstantinos Hatzistergos ◽  
Raul Dulce ◽  
Amarylis Wanschel ◽  
Wayne Balkan ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Signaling Pathways ◽  
Signaling Pathway ◽  
Es Cells ◽  
Embryoid Bodies ◽  
Negative Regulator ◽  
Cell Based Therapy ◽  
Cardiac Phenotype ◽  
Cardiac Mesoderm

Introduction: The mechanism by which signaling pathways, such as Wnt and BMP interact and modulate each other’s function is crucial to our understanding of cardiomyogenesis and cardiomyocyte proliferation. Nitric oxide (NO) is a signaling molecule that can trigger cardiac differentiation of stem cells, suggesting a cardiogenic function of NO synthase(s) (NOS). Hypothesis: NO modulates transcription factor function during pluripotency and differentiation toward a cardiac phenotype. Methods: Induced pluripotent stem cells (iPSCs) were derived from fibroblasts from wildtype mice and mice lacking S-nitrosoglutathione reductase (GSNOR -/- ), a denitrosylase that regulates protein S-nitrosylation. iPSCs were differentiated into functional cardiomyocytes from embryoid bodies (EBs) via the hanging-drop method. Results: During differentiation into cardiomyocytes, GSNOR -/- iPSC-derived cardiomyocytes exhibited reduced expression of mesoderm induction-related ( Brachyury ), cardiac mesoderm (Kdr , Isl-1 ) and cardiac progenitor genes ( Nkx2.5 , GATA4 ). Axin-1, an inducer of apoptosis and negative regulator of the Wnt signaling pathway and MAPK pathways, specifically p38, were increased on EB-Day (D)4. In contrast, SMAD1/5/8, members of the BMP canonical signaling pathway, were reduced beginning on EB-D8. Increased p38 is associated with reduced GATA4 expression and differentiation of human ES cells into cardiomyocytes. Decreased SMAD1/5/8 is likely at least in part responsible for the reduced expression of Nkx2.5. Conclusions: Our findings support that the absence of GSNOR modulates Wnt/β-catenin and BMP signaling pathways during cardiogenesis, resulting in reduced expression of mesoderm, cardiac mesoderm and cardiac progenitor genes. These findings are expected to have important implications for regenerative medicine and can provide new targets for iPS cell-based therapy.

scholarly journals Microarray analysis identification of key pathways and interaction network of differential gene expressions during osteogenic differentiation

2020 ◽  
Vol 14 (1) ◽  
Author(s):  
Fatemeh Khodabandehloo ◽  
Sara Taleahmad ◽  
Reza Aflatoonian ◽  
Farzad Rajaei ◽  
Zahra Zandieh ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Signaling Pathway ◽  
Expression Profiles ◽  
Wnt Signaling Pathway ◽  
Hub Genes ◽  
Gene Expressions ◽  
Cell Based Therapy ◽  
Wnt Pathways ◽  
Key Pathways

Abstract Background Adult bone marrow-derived mesenchymal stem cells (BM-MSCs) are multipotent stem cells that can differentiate into three lineages. They are suitable sources for cell-based therapy and regenerative medicine applications. This study aims to evaluate the hub genes and key pathways of differentially expressed genes (DEGs) related to osteogenesis by bioinformatics analysis in three different days. The DEGs were derived from the three different days compared with day 0. Results Gene expression profiles of GSE37558 were obtained from the Gene Expression Omnibus (GEO) database. A total of 4076 DEGs were acquired on days 8, 12, and 25. Gene ontology (GO) enrichment analysis showed that the non-canonical Wnt signaling pathway and lipopolysaccharide (LPS)-mediated signaling pathway were commonly upregulated DEGs for all 3 days. KEGG pathway analysis indicated that the PI3K-Akt and focal adhesion were also commonly upregulated DEGs for all 3 days. Ten hub genes were identified by CytoHubba on days 8, 12, and 25. Then, we focused on the association of these hub genes with the Wnt pathways that had been enriched from the protein-protein interaction (PPI) by the Cytoscape plugin MCODE. Conclusions These findings suggested further insights into the roles of the PI3K/AKT and Wnt pathways and their association with osteogenesis. In addition, the stem cell microenvironment via growth factors, extracellular matrix (ECM), IGF1, IGF2, LPS, and Wnt most likely affect osteogenesis by PI3K/AKT.

Abstract 19282: Transient Bone Morphogenic Protein Antagonism Directs Differentiation of Ipscs Into the Cardiac Neural Crest and Ckit+ Myocardial Progenitor Lineages

Circulation ◽  
2015 ◽  
Vol 132 (suppl_3) ◽  
Author(s):  
Konstantinos E Hatzistergos ◽  
Lauro M Takeuchi ◽  
Dieter Saur ◽  
Barbara Seidler ◽  
Susan M Dymecki ◽  
...  
Keyword(s):  
Neural Crest ◽  
Signaling Pathway ◽  
Progenitor Cell ◽  
Embryoid Bodies ◽  
Cardiac Neural Crest ◽  
Specific Induction ◽  
Cre Recombination ◽  
Bmp Antagonist ◽  
Cardiogenic Differentiation ◽  
Cardiac Mesoderm

Introduction: The signaling pathways that govern cKit+ cardiac progenitor cell (CPC) differentiation into cardiomyocytes (CMs) are unknown. Some studies suggest an essential role in cardiomyogenesis, others suggest a minimal CPC contribution. We studied if it is a non-permissive cardiac milieu that minimizes the generation of CMs from CPCs. Hypothesis: Transient BMP antagonism directs the generation of cardiomyocytes from CPCs. Methods: We lineage-traced CPCs using a novel dual-recombinase responsive indicator mice (cKitCreERT2;Wnt1::Flpe;RC::Fela) and iPSCs derived from cKitCreERT2;IRG (iPSCKit) mice. Results: Intersectional genetic fate-mapping of cKitCreERT2;Wnt1:: Flpe;RC::Fela embryos supported that cKit marks Wnt1-expressing cardiac neural crest (CNC) progenitors, emerging at ~E9.5 and contributing a limited number of cardiomyocytes (n=3). We lineage-traced CPCs during stage-specific cardiogenic differentiation of iPSCKit. Ascorbate treatment promoted differentiation of iPSCKit-derived embryoid bodies (EBs) into Nkx2.5+ myocardium, 45.5%±6.7% of which co-expressed the Cre-reporter EGFP (n=154 EBs; 12 preps), suggesting that CPCs encompass fully competent cardiomyogenic progenitors. Noggin (or Dorsomorphin), a BMP antagonist transiently expressed in the heart at E7.5-E8.5 but not during CNC invasion, directed the differentiation of iPSCkit-EBs into Mesp1+/Isl1+/Nkx2.5+ cardiac mesoderm progenitors (p≤0.0001). The same signaling pathway subsequently directed EBs into the cKit+/Wnt1+/Pax3+/Mitf-H+/Isl1+/Nkx2.5+ CNC lineage (p≤0.0001), while suppressing the generation of WT1+/Tbx18+ epicardium (p<0.05). Stage-specific induction of Cre-recombination delineated that iPSCkit-derived CPCs encompass Mesp1–/cKit+/Nkx2.5+ CNC progenitors, which contributed EGFP+ CNC derivatives, including Nkx2-5+ cardiomyocytes, to 60.7%±7.3% of spontaneously beating EBs (n=147 EBs; 12 preps). Conclusions: Our data show that CPCkit are fully competent CNC-derived cardiomyogenic progenitors, whose differentiation to cardiomyocytes is minimized by a latent Noggin-mediated signaling pathway. Therapeutically exploiting CPCkit, provides an important strategy for maximizing myocardial regeneration.

Overexpression of HOXB4 enhances the hematopoietic potential of embryonic stem cells differentiated in vitro

Blood ◽  
1996 ◽  
Vol 87 (7) ◽  
pp. 2740-2749 ◽  
Author(s):  
CD Helgason ◽  
G Sauvageau ◽  
HJ Lawrence ◽  
C Largman ◽  
RK Humphries
Keyword(s):  
Stem Cells ◽  
Es Cells ◽  
Hematopoietic Cells ◽  
Embryonic Stem ◽  
Homeobox Genes ◽  
Embryoid Bodies ◽  
Proliferative Potential ◽  
Hematopoietic Stem ◽  
Differentiation And Proliferation

Little is known about the molecular mechanisms controlling primitive hematopoietic stem cells, especially during embryogenesis. Homeobox genes encode a family of transcription factors that have gained increasing attention as master regulators of developmental processes and recently have been implicated in the differentiation and proliferation of hematopoietic cells. Several Hox homeobox genes are now known to be differentially expressed in various subpopulations of human hematopoietic cells and one such gene, HOXB4, has recently been shown to positively determine the proliferative potential of primitive murine bone marrow cells, including cells with long-term repopulating ability. To determine if this gene might influence hematopoiesis at the earliest stages of development, embryonic stem (ES) cells were genetically modified by retroviral gene transfer to overexpress HOXB4 and the effect on their in vitro differentiation was examined. HOXB4 overexpression significantly increased the number of progenitors of mixed erythroid/myeloid colonies and definitive, but not primitive, erythroid colonies derived from embryoid bodies (EBs) at various stages after induction of differentiation. There appeared to be no significant effect on the generation of granulocytic or monocytic progenitors, nor on the efficiency of EB formation or growth rate. Analysis of mRNA from EBs derived from HOXB4-transduced ES cells on different days of primary differentiation showed a significant increase in adult beta-globin expression, with no detectable effect on GATA-1 or embryonic globin (beta H-1). Thus, HOXB4 enhances the erythropoietic, and possibly more primitive, hematopoietic differentiative potential of ES cells. These results provide new evidence implicating Hox genes in the control of very early stages in the development of the hematopoietic system and highlight the utility of the ES model for gaining insights into the molecular genetic regulation of differentiation and proliferation events.

scholarly journals Generation of a nitric oxide signaling pathway in mesenchymal stem cells promotes endothelial lineage commitment

2019 ◽  
Vol 234 (11) ◽  
pp. 20392-20407 ◽  
Author(s):  
Nadeeka Bandara ◽  
Saliya Gurusinghe ◽  
Anne Kong ◽  
Geraldine Mitchell ◽  
Le‐Xin Wang ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Signaling Pathway ◽  
Lineage Commitment ◽  
Nitric Oxide Signaling ◽  
Endothelial Lineage

Filamin A Controls Embryonic Stem Cell Differentiation.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4599-4599
Author(s):  
Taisuke Kanaji ◽  
Takashi Okamura ◽  
Peter J. Newman
Keyword(s):  
Cell Differentiation ◽  
Signaling Pathways ◽  
Es Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Embryoid Bodies ◽  
Actin Binding ◽  
Embryonic Stem Cell Differentiation ◽  
Filamin A ◽  
Es Cell

Abstract Abstract 4599 Filamin A is a major non-muscle actin binding protein that plays an important role in cross-linking cortical actin filaments into three-dimensional networks. In addition to its role as a cytoskeletal scaffolding molecule, Filamin A is also known to bind more than 30 other proteins, regulating their subcellular location and coordinating their ability to signal. To analyze the role of filamin A in mouse embryonic stem (ES) cell maturation, we generated filamin ALow ES cells by introducing a micro-RNA that specifically downregulates filamin A expression under the control of a cytomegalovirus promoter. Filamin ALow ES cells exhibited a more rounded morphology than did their wild-type filamin ANormal counterparts, and expressed increased levels of the ES cell transcription factor Nanog. In contrast, non-transfected cells in the same culture dish retained normal expression of filamin A, expressed low levels of Nanog, and exhibited a more elongated and spread phenotype characteristic of differentiating cells. Further evidence for a role for filamin A in ES cell differentiation was provided by the observation that withdrawing leukemia inhibitory factor (LIF) to induce ES cell differentiation was accompanied by increased expression of filamin A, a concomitant loss of Nanog expression, and acquisition of a differentiated morphology. Filamin ALow ES cells were able to retain their undifferentiated phenotype, as evaluated by alkaline phosphatase (Alp) activity, in the presence of a 10-fold lower concentration of LIF than was permissive for filamin ANormal ES cells, or following exposure to the differentiating agent, bone morphogenic protein 4 (BMP4). LIF-induced phosphorylation of ERK was decreased in filamin ALow relative to filamin ANormal ES cells, as was BMP-induced phosphorylation of Smad1/5 - two signaling pathways that initiate ES cell differentiation. Finally, embryoid bodies comprised of filamin ALow ES cells were unable to differentiate into CD41+ hematopoietic progenitor cells. Taken together, these data demonstrate that filamin A plays a previously unrecognized, but critical, scaffolding function that support both the LIF - ERK and BMP4 - Smad1/5 signaling pathways leading to ES and hematopoietic cell differentiation. Manipulation of filamin levels might be useful in the future to modulate the differentiation requirements for a variety of clinically-and therapeutically-useful stem cells. Disclosures: Newman: Novo Nordisk: Consultancy; New York Blood Center: Membership on an entity's Board of Directors or advisory committees.

Stimulation of Cardiomyogenesis of Embryonic Stem Cells by Nitric Oxide Downstream of AMP-Activated Protein Kinase and mTOR Signaling Pathways

2011 ◽  
Vol 20 (12) ◽  
pp. 2163-2175 ◽  
Author(s):  
Manju Padmasekar ◽  
Fatemeh Sharifpanah ◽  
Andreas Finkensieper ◽  
Maria Wartenberg ◽  
Heinrich Sauer
Keyword(s):  
Nitric Oxide ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Protein Kinase ◽  
Signaling Pathways ◽  
Embryonic Stem ◽  
Mtor Signaling ◽  
Amp Activated Protein Kinase ◽  
Stimulation Of

Induction of Cardiomyocyte Differentiation From Mouse Embryonic Stem Cells in a Confined Microfluidic Environment

2009 ◽  
Author(s):  
Chen-rei Wan ◽  
Seok Chung ◽  
Ryo Sudo ◽  
Roger D. Kamm
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Mechanical Stretch ◽  
Embryoid Bodies ◽  
Negative Regulator ◽  
Differentiation Process ◽  
Cardiomyocyte Differentiation

Embryonic stem cell derived cardiomyocytes are deemed an attractive treatment option for myocardial infarction. Their clinical efficacy, however, has not been unequivocally demonstrated. There is a need for better understanding and characterization of the cardiogenesis process. A microfluidic platform in vitro is used to dissect and better understand the differentiation process. Through this study, we find that while embryoid bodies (EBs) flatten out in a well plate system, differentiated EBs self-assemble into complex 3D structures. The beating regions of EBs are also different. Most beating areas are observed in a ring pattern on 2D well plates around the center, self-assembled beating large 3D aggregates are found in microfluidic devices. Furthermore, inspired by the natural mechanical environment of the heart, we applied uniaxial cyclic mechanical stretch to EBs. Results suggest that prolonged mechanical stimulation acts as a negative regulator of cardiogenesis. From this study, we conclude that the culture environments can influence differentiation of embryonic stem cells into cardiomycytes, and that the use of microfluidic systems can provide new insights into the differentiation process.

scholarly journals 177 GENERATION OF ES CELLS AND TRANSGENIC MICE EXPRESSING MTERT-GFP AS A MARKER OF PLURIPOTENTIAL CELLS

2005 ◽  
Vol 17 (2) ◽  
pp. 239
Author(s):  
M.A. Ramírez ◽  
R. Fernández-González ◽  
P.N. Moreira ◽  
M. Pérez-Crespo ◽  
J. de la Fuente ◽  
...  
Keyword(s):  
Stem Cells ◽  
Transgenic Mice ◽  
Fluorescent Protein ◽  
Es Cells ◽  
Promoter Sequence ◽  
Telomerase Activity ◽  
Embryoid Bodies ◽  
Gfp Expression ◽  
Adult Tissues ◽  
Different Levels

There is not a simple system that allows us to identify stem cells in adult tissues. Cells of adult tissues arise from dividing progenitor cells, which themselves are derived from multipotential stem cells. Telomerase is the enzyme that maintains the ends of linear chromosomes in eukaryotic cells. Recently, a segment of the promoter sequence of the reverse transcriptase of murine telomerase (mTert) has been characterized. mTert is expressed with greatest abundance during embryogenesis and becomes widely expressed in adult tissues at low levels. This low expression level in adult tissues may be due to the presence of pluripotent stem cells present in those tissues. To examine the relationship between telomerase activity and multipotential of adult cells we have generated three constructs (1k-, 2k-, and 5k-mTert-GFP) comprising different segments of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (GFP). These constructs were electroporated into R1 and B6D2 (generated in our laboratory) ES cells and were used to produce transgenic mice. The generation and identification of transgenic mice (C57BL6 × CBA) has been previously described (Gutierrez-Adan and Pintado 2000 Trangenic Res. 9, 81–89). Transgenic founders were backcrossed to C57BL6 × CBA mice to obtain transgenic lines. The three constructs were able to mimic the mTert expression, which was coupled to green fluorescence. The mTert-GFP transfected ES cells were initially maintained in medium supplemented with LIF, which was subsequently removed to allow differentiation of embryoid bodies (EBs) and other cell types. GFP expression was higher during the first two days after LIF removal (period of enhanced cell proliferation), decreasing in the following days as a result of EB differentiation. Both ES cell lines showed reduced GFP expression upon differentiation, suggesting that mTert is the principal determinant of telomerase activity; moreover, different degrees of expression and down regulation were reported with the different constructs. Using these constructs we have also generated transgenic mice. Eight lines of transgenic mice carrying the 1kmTert-GFP transgene, four with the 2kmTert-GFP, and three with the 5kmTert-GFP, were obtained. There were no significant differences between the proportions of transgenic founder generates. The transgenic mice express and GFP during the fetal development, indicating their telomerase activity. We are now analyzing the expression of mTert-GFP in adults tissues. Our results suggest that telomerase-GFP transgenics are an important tool to assess the role of telomerase in adult multipotential cells as well as to select these pluripotent cells in adult tissue. It will be interesting to see if different levels of mTert-GFP expression are associated with different levels of pluripotency.

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.

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