scholarly journals Assessment of the Potential of CDK2 Inhibitor NU6140 to Influence the Expression of Pluripotency Markers NANOG, OCT4, and SOX2 in 2102Ep and H9 Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-16 ◽  
Author(s):  
Ade Kallas ◽  
Martin Pook ◽  
Annika Trei ◽  
Toivo Maimets
Keyword(s):  
Cell Cycle Progression ◽  
Single Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Flow Cytometric Method ◽  
Histone 3 ◽  
Flow Cytometric ◽  
M Phase ◽  
Induced Apoptosis ◽  
Hes Cells

As cyclin-dependent kinases (CDKs) regulate cell cycle progression and RNA transcription, CDKs are attractive targets for creating cancer cell treatments. In this study we investigated the effects of the small molecular agent NU6140 (inhibits CDK2 and cyclin A interaction) on human embryonic stem (hES) cells and embryonal carcinoma-derived (hEC) cells via the expression of transcription factors responsible for pluripotency. A multiparameter flow cytometric method was used to follow changes in the expression of NANOG, OCT4, and SOX2 together in single cells. Both hES and hEC cells responded to NU6140 treatment by induced apoptosis and a decreased expression of NANOG, OCT4, and SOX2 in surviving cells. A higher sensitivity to NU6140 application in hES than hEC cells was detected. NU6140 treatment arrested hES and hEC cells in the G2 phase and inhibited entry into the M phase as evidenced by no significant increase in histone 3 phosphorylation. When embryoid bodies (EBs) formed from NU6104 treated hES cells were compared to EBs from untreated hES cells differences in ectodermal, endodermal, and mesodermal lineages were found. The results of this study highlight the importance of CDK2 activity in maintaining pluripotency of hES and hEC cells and in differentiation of hES cells.

scholarly journals Bnip3 and AIF cooperate to induce apoptosis and cavitation during epithelial morphogenesis

2012 ◽  
Vol 198 (1) ◽  
pp. 103-114 ◽  
Author(s):  
Yanmei Qi ◽  
Xiaoxiang Tian ◽  
Jie Liu ◽  
Yaling Han ◽  
Alan M. Graham ◽  
...  
Keyword(s):  
Protein A ◽  
Hypoxia Inducible Factor ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Epithelial Morphogenesis ◽  
Dependent Manner ◽  
Interacting Protein ◽  
The Core ◽  
Adenovirus E1b ◽  
Induced Apoptosis

Apoptosis is an essential step in cavitation during embryonic epithelial morphogenesis, but its mechanisms are largely unknown. In this paper, we used embryonic stem cell–differentiated embryoid bodies (EBs) as a model and found that Bnip3 (Bcl-2/adenovirus E1B 19-kD interacting protein), a BH3-only proapoptotic protein, was highly up-regulated during cavitation in a hypoxia-dependent manner. Short hairpin RNA silencing of Bnip3 inhibited apoptosis of the core cells and delayed cavitation. We show that the Bnip3 up-regulation was mediated mainly by hypoxia-inducible factor (HIF)–2. Ablation of HIF-2α or HIF-1β, the common β subunit of HIF-1 and -2, suppressed Bnip3 up-regulation and inhibited apoptosis and cavitation. We further show that apoptosis-inducing factor (AIF) cooperated with Bnip3 to promote lumen clearance. Bnip3 silencing in AIF-null EBs nearly blocked apoptosis and cavitation. Moreover, AIF also regulated Bnip3 expression through mitochondrial production of reactive oxygen species and consequent HIF-2α stabilization. These results uncover a mechanism of cavitation through hypoxia-induced apoptosis of the core cells mediated by HIFs, Bnip3, and AIF.

scholarly journals Cell Cycle Modulation of CHO-K1 Cells Under Genistein Treatment Correlates with Cells Senescence, Apoptosis and ROS Level but in a Dose-Dependent Manner

2019 ◽  
Vol 9 (3) ◽  
pp. 453-461
Author(s):  
Riris Istighfari Jenie ◽  
Nur Dina Amalina ◽  
Gagas Pradani Nur Ilmawati ◽  
Rohmad Yudi Utomo ◽  
Muthi Ikawati ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Progression ◽  
Cell Senescence ◽  
Dependent Manner ◽  
Protein Markers ◽  
High Concentration ◽  
Physiological Concentration ◽  
Genistein Treatment ◽  
M Phase ◽  
Induced Apoptosis

Purpose: Genistein, a soy isoflavone, exhibits a biphasic effect on cells proliferation with some different effects between ER-alpha and ER-beta. The objective of this present study is to determine the modulatory effect based on cell cycle progression under genistein treatment in combination with 17-β estradiol (E2) on CHO-K1 cells. Methods: The effect of genistein 0.1-100 µM on cells proliferation was examined by MTT assay. The modulation of genistein and estradiol (E2) on cell cycle and apoptosis were observed by using flowcytometry with PI and PI/AnnexinV staining, respectively. Moreover, the effect of genistein and E2 on senescence cells, and ROS level were determined by senescence-associated β-galactosidase (SA β-gal) staining and by using flowcytometry with 2’, 7’–dichlorofluorescin diacetate (DCFDA) staining, respectively. The expression level of the cell cycle and senescence protein markers were observed by immunoblotting. Results: Single treatment of genistein at physiologically achievable (low) concentration (<2 µM) induced proliferation of CHO-K1 cells while at a pharmacological (high) concentration (50 and 100 µM) suppressed cells proliferation. Interestingly, treatment of genistein at the physiological concentration in combination with E2 for 24, 48 and 72 h decreased cells viability on CHO-K1 cells compared to untreated cells. Further analysis of the cells showed that 50 µM genistein induced G2/M phase accumulation and induced apoptosis. Moreover, genistein induced cell senescence and increased ROS level. Immunoblotting analysis showed the decreasing of ERalpha, Bcl2, and ppRb protein level upon treatment of 1 µM Gen and 1 nM E2. Conclusion: Our results suggest that the cell proliferation inhibitory mechanism of genistein at pharmacological concentration involved the induction of cell senescence, and the elevation of ROS level. Moreover, the decreased of cells proliferation upon treatment of physiological concentration of genistein in combination with E2 may be correlated with the alteration of ER expression.

scholarly journals Single cell profiling of total RNA using Smart-seq-total

2020 ◽  
Author(s):  
Alina Isakova ◽  
Norma Neff ◽  
Stephen R. Quake
Keyword(s):  
Single Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
High Yield ◽  
Rna Seq ◽  
Mcf7 Cells ◽  
Total Rna ◽  
Non Coding Rna

ABSTRACTThe ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current RNA-seq methods are unable to simultaneously monitor both short and long, poly(A)+ and poly(A)-transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here, we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and non-coding RNA from a single cell. Built upon the template-switch mechanism, Smart-seq-total bears the key feature of its predecessor, Smart-seq2, namely, the ability to capture full-length transcripts with high yield and quality. It also outperforms current poly(A)–independent total RNA-seq protocols by capturing transcripts of a broad size range, thus, allowing us to simultaneously analyze protein-coding, long non-coding, microRNA and other non-coding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T and MCF7 cells as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. We show that simultaneous measurement of non-coding RNA and mRNA from the same cell enables elucidation of new roles of non-coding RNA throughout essential processes such as cell cycle or lineage commitment. Moreover, we show that cell types can be distinguished based on the abundance of non-coding transcripts alone.

scholarly journals Single-cell quantification of a broad RNA spectrum reveals unique noncoding patterns associated with cell types and states

2021 ◽  
Vol 118 (51) ◽  
pp. e2113568118
Author(s):  
Alina Isakova ◽  
Norma Neff ◽  
Stephen R. Quake
Keyword(s):  
Single Cell ◽  
Noncoding Rna ◽  
Single Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
Embryoid Bodies ◽  
Rna Seq ◽  
Total Rna ◽  
Rna Transcripts ◽  
Rna Content

The ability to interrogate total RNA content of single cells would enable better mapping of the transcriptional logic behind emerging cell types and states. However, current single-cell RNA-sequencing (RNA-seq) methods are unable to simultaneously monitor all forms of RNA transcripts at the single-cell level, and thus deliver only a partial snapshot of the cellular RNAome. Here we describe Smart-seq-total, a method capable of assaying a broad spectrum of coding and noncoding RNA from a single cell. Smart-seq-total does not require splitting the RNA content of a cell and allows the incorporation of unique molecular identifiers into short and long RNA molecules for absolute quantification. It outperforms current poly(A)-independent total RNA-seq protocols by capturing transcripts of a broad size range, thus enabling simultaneous analysis of protein-coding, long-noncoding, microRNA, and other noncoding RNA transcripts from single cells. We used Smart-seq-total to analyze the total RNAome of human primary fibroblasts, HEK293T, and MCF7 cells, as well as that of induced murine embryonic stem cells differentiated into embryoid bodies. By analyzing the coexpression patterns of both noncoding RNA and mRNA from the same cell, we were able to discover new roles of noncoding RNA throughout essential processes, such as cell cycle and lineage commitment during embryonic development. Moreover, we show that independent classes of short-noncoding RNA can be used to determine cell-type identity.

scholarly journals Hepatocyte Differentiation from Human ES Cells using the Simple Embryoid Body Formation Method and the Staged-Additional Cocktail

2009 ◽  
Vol 9 ◽  
pp. 884-890 ◽  
Author(s):  
Katsunori Sasaki ◽  
Hinako Ichikawa ◽  
Shunsuke Takei ◽  
Hee Sung No ◽  
Daihachiro Tomotsune ◽  
...  
Keyword(s):  
Embryoid Body ◽  
Es Cells ◽  
Embryonic Stem ◽  
Culture Method ◽  
Polymerase Chain Reaction Analysis ◽  
Embryoid Bodies ◽  
Hepatocyte Differentiation ◽  
Gene Markers ◽  
Transmission Electron ◽  
Hes Cells

To induce hepatocytes from human embryonic stem (hES) cells easily and effectively, a simple suspension culture method that separates ES colonies with a scraper and transfers them into newly developed, nonadherent MPC (2-methacryloyloxyethyl phosphorylcholine) plates, and the staged-additional cocktail method, including growth factors, cytokines, and Lanford serum-free medium, were developed and evaluated mainly by morphological analysis. The formed embryoid bodies (EBs) showed compact cellular agglomeration until day 4 and later formed coeloms in their interior. RT-PCR (reverse transcriptase-polymerase chain reaction) analysis showed that they are gene markers of the three germ layers. Mesenchymal cells with rough endoplasmic reticulum (rER) and extracellular matrix (ECM), and without junctions, were recognized in the interior of the EBs by transmission electron microscopy (TEM) in addition to epithelial cells. When they were stimulated by the staged-additional cocktail, they expressed albumin-positive immunoreactivity, indocyanine green (ICG) uptake, and typical ultrastructures of the hepatocytes, including bile canaliculi. These results indicate that these combined methods promote EB formation and hepatocyte differentiation from hES cells.

scholarly journals 12-Deoxyphorbol Esters Induce Growth Arrest and Apoptosis in Human Lung Cancer A549 Cells Via Activation of PKC-δ/PKD/ERK Signaling Pathway

2020 ◽  
Vol 21 (20) ◽  
pp. 7579
Author(s):  
Ju-Ying Tsai ◽  
Dóra Rédei ◽  
Judit Hohmann ◽  
Chin-Chung Wu
Keyword(s):  
Lung Cancer ◽  
Anticancer Agents ◽  
Cell Cycle Progression ◽  
Nuclear Translocation ◽  
Growth Arrest ◽  
A549 Cells ◽  
Cell Types ◽  
Human Lung Cancer ◽  
M Phase ◽  
Induced Apoptosis

Prostratin, a non-tumor promoting 12-deoxyphorbol ester, has been reported as a protein kinase C (PKC) activator and is shown to have anti-proliferative activity in certain cancer cell types. Here we show that GRC-2, a prostratin analogue isolated from Euphorbia grandicornis, is ten-fold more potent than prostratin for inhibiting the growth of human non-small cell lung cancer (NSCLC) A549 cells. Flow cytometry assay revealed that GRC-2 and prostratin inhibited cell cycle progression at the G2/M phase and induced apoptosis. The cytotoxic effect of GRC-2 and prostratin was accompanied by activation and nuclear translocation of PKC-δ and PKD as well as hyperactivation of extracellular signal-related kinase (ERK). Knockdown of either PKC-δ, PKD or ERK significantly protected A549 cancer cells from GRC-2- and prostratin-induced growth arrest as well as apoptosis. Taken together, our results have shown that prostratin and a more potent analogue GRC-2 reduce cell viability in NSCLC A549 cells, at least in part, through activation of the PKC-δ/PKD/ERK pathway, suggesting the potential of prostratin and GRC-2 as anticancer agents.

A murine respiratory-inducing niche displays variable efficiency across human and mouse embryonic stem cell species

2007 ◽  
Vol 292 (5) ◽  
pp. L1241-L1247 ◽  
Author(s):  
Mark Denham ◽  
Brock J. Conley ◽  
Fredrik Olsson ◽  
Lerna Gulluyan ◽  
Timothy J. Cole ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Single Cells ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies ◽  
Mouse Lung ◽  
Specific Marker ◽  
Tubule Formation ◽  
Human Specific

Human embryonic stemlike cells (hESCs) are pluripotent cells derived from blastocysts. Differentiating hESCs into respiratory lineages may benefit respiratory therapeutic programs. We previously demonstrated that 24% of all mouse embryonic stem cell (mESC) derivatives cocultured with embryonic day 11.5 (E11.5) mouse lung rudiments display immunoreactivity to the pneumonocyte II specific marker surfactant-associated protein C (Sftpc). Here we further investigate the effects of this inductive niche in terms of its competence to induce hESC derivative SFTPC immunoreactivity and the expression of other markers of terminal lung secretory units. When hESCs were cocultured as single cells, clumps of ∼10 cells or embryoid bodies (EBs), hESC derivatives formed pan-keratin-positive epithelial tubules at high frequency (>30% of all hESC derivatives). However, human-specific SFTPC immunoreactivity associated with tubule formation only at low frequency (<0.1% of all hESC derivatives). Human-specific SFTPD and secretoglobin family 1A member 1 ( SCGB1A1, also known as CC10) transcripts were detected by PCR after prolonged culture. Expression of other terminal lung secretory unit markers ( TITF1, SFTPA, and SFTPB) was not detected at any time point analyzed. On the other hand, hESC derivatives cultured as plated EBs in media previously demonstrated to induce Sftpc expression in isolated mouse fetal tracheal epithelium expressed all terminal lung secretory unit markers examined. mESCs and hESCs thus display fundamental differences in their response to the E11.5 mouse lung inductive niche, and these data provide an important step in the delineation of signaling mechanisms capable of efficiently inducing hESC differentiation into terminal secretory units of the lung.

Myogenic Developmental Potential of Hemangioblasts Derived from Mouse Embryonic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1702-1702
Author(s):  
Michael J. Burke ◽  
Patrick Kennedy ◽  
Lei Wang ◽  
Devang Bhatt ◽  
William T. Tse
Keyword(s):  
Skeletal Muscle ◽  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Muscle Cells ◽  
Mouse Embryonic Stem Cells ◽  
Embryoid Bodies ◽  
C2c12 Cells ◽  
Developmental Potential

Abstract The hemangioblast, the progenitor common to hematopoetic and endothelial lineages, expresses the vascular endothelial growth factor (VEGF) receptor flk-1 on its surface. Based on lineage-tracking studies indicating that skeletal muscle cells are derived from flk-1-expressing precursors (Sato et al. Genesis35:153–159, 2003), we tested the hypothesis that the hemangioblast is also a progenitor to the muscle lineage. We studied the development of skeletal muscle progentiors from mouse embryonic stem cells cultured in suspension to form embryoid bodies (EB). These EB were harvested at different time points, disaggregated into single cells and analyzed by flow cytometry. After 10–18 days of culture, the EB contained abundant cells that expressed α7-integrin, a skeletal muscle-specific cell surface antigen. Immunofluorescence microscopic study of sorted and cytospun α7-integrin-positive cells from EB revealed positive staining with MyoD, sarcomeric α-actinin and desmin antibodies, indicating that they were bona fide muscle cells. To test if these muscle cells were derived from hemangioblasts, green fluorescent protein (GFP)-expressing EB were collected at day 4 of development, disaggregated into single cells and replated in methylcellulose medium with the addition of VEGF, stem cell factor and thrombopoietin. After 5 days in methylcellulose culture, clusters of loosely aggregated cells appeared. These cells have features characteristic of hemangioblasts and exhibit large nuclear-to-cytoplasmic ratios. The hemangioblast colonies were individually picked, diaggregated into single cells and plated with a myoblastic cell line (C2C12 cells) in media containing 10% fetal calf serum. Co-culturing of the hemangioblasts with a myoblastic feeder layer was designed to provide the environmental signals necessary for myogenic development. After 48 hours, the co-culture medium was changed to 2% serum to promote muscle differentiation. Three days later, the plates were fixed and analyzed. A small number of GFP-expressing cells assumed an elongated, multinucleated, myofiber-like appearance and stained positiviely for skeletal muscle markers α7-integrin and desmin. We next tested the myogenic potential of hemangioblasts in vivo. The tibialis anterior muscles of C57BL/6 mice were injected with cardiotoxin, a protein kinase inhibitor that depolarizes muscle fibers and induces muscle regeneration. After two days, hemangioblast colonies were picked from methylcellulose culture and injected into the regenerating muscles. Occasional GFP-expressing fibers that stained positive for desmin were seen when analyzed several weeks post-injection. These results indicate that the hemangioblast may be a progenitor not only to hematopoetic and endothelial lineages, but also to skeletal muscle. Future experiments include investigating the significance and mechanism of the hemangioblast’s development into muscle and explore the role of cell-cell fusion in this phenomenon.

Charaterization of Human Primitive Megakaryocytes and “Platelets”.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1520-1520
Author(s):  
Dong Chen ◽  
Rachel L. Lewis ◽  
Christine A. Daigh ◽  
James A. Thomson
Keyword(s):  
Bone Marrow ◽  
Embryonic Stem ◽  
Coagulation Factors ◽  
Preliminary Evidence ◽  
Embryoid Bodies ◽  
Hla Dr ◽  
Functional Aspects ◽  
Functional Features ◽  
Beta 2 Microglobulin ◽  
Hes Cells

Abstract Primitive megakaryocytes (PMs) have been first observed in murine embroynal yolk sac [Xu et al. Blood. 2001 Apr 1;97(7):2016–22.]. The biological characteristics and functional aspects of human primitive megakaryocytes are not well established even though they can be transiently observed by bone marrow stromal cell (OP9) co-culture methodology [Gaur, M et al J Thromb Haemost.2006 Feb;4(2):436–42.; Eto K et al. Proc Natl Acad Sci U S A.2002 Oct 1;99(20):12819–24.]. The goal of this study is to charaterize PMs’ unique biological and functional features. To obtain a pure population of human primitive megakaryocytes, human embryonic stem (hES) cells were cultured to form embryoid bodies. CD34+/CD31+/VE-Cadherin+/Flk-1+ hemangioblasts on day 12 were isolated and further cultured for 10–12 days in media containing thrombopoietin (TPO), interleukin 3 (IL3), stem cell factor (SCF). 30% of the non-adherent cells were strongly positive for CD31/CD41/CD61/CD42b, and weakly positive for CD42a/CD45/beta-2-microglobulin (B2M) but negative for CD117 or HLA-DR. The DNA ploidies are exclusively 2N–8N confirming the primitive status of PMs. Morphologically when compared to bone marrow derived megakaryocytes, PMs readily produce long processes with chains of “proplatelet” like structures. By live videomicroscopy, these “proplatelet” structures can actively recruit mitochondria. Interestingly, “platelet-like” fragment shedding from megakaryocytes is significantly stimulated in the presence of nitric oxide and human plasma. The “platelet-like” fragments and megakaryocytes respond to strong agonists, including thrombin and TRAP, with aggregation, alpha granule release and increased affinity for fibrinogen. In contrast, weaker agonists such as ADP only generate minimal response. This study provides preliminary evidence that primitive megakaryocytes have acquired some basic machinery of hemostasis even when the blood flow, liver function and coagulation factors are not present at the early phase of embryonal development. [We thank Dr.William L. Nichols at Mayo Clinic for his critical reviews of this abstract.]

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