Abstract P023: HDAC Inhibition Promotes Cardiogenesis and the Survival of Embryonic Stem Cells Through Proteasome-Dependent Pathway

2011 ◽  
Vol 109 (suppl_1) ◽  
Author(s):  
Ting C Zhao ◽  
Hongping Chen ◽  
Megan DeNicola ◽  
Yu Zhao ◽  
Xin Qin ◽  
...  
Keyword(s):  
Cell Death ◽  
Sodium Butyrate ◽  
Hdac Inhibitors ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Luciferase Assay ◽  
Hdac Inhibition ◽  
Cardiac Lineage ◽  
Proteasome Pathway ◽  
The Impact

Objectives: Histone deacetylase (HDAC) inhibition plays a crucial role in mediating cardiogenesis and myocardial protection, whereas HDAC degradation has recently attracted attention in mediating the biological function of HDACs. However, it remains unknown whether HDAC inhibition modulates cardiogenesis and embryonic stem cell (ESC) survival through the proteasome pathway. Methods and Results: Using the well-established mouse CGR8 mouse ESC culture, we evaluated the impact of HDAC inhibition and proteasome pathway on the cell death, viability and apoptosis in ESCs in response to oxidant stress (100μ mol/L hydrogen peroxides). We demonstrated that HDAC inhibitors, both TSA (50 nmol/L) and sodium butyrate (200 μ mol/L) that causes the pronounced reduction of HDAC4 activity, decreased cell death and increased viability of ESCs. HDAC inhibition reduced the cleaved caspase 3, 6, 9, PARP and TUNE-positive ESCs, which were abrogated with MG132 (0.5 μ mol/L), a specific proteasome inhibitor. Furthermore, we employed in vitro “hanging drop” methods to carry out two weeks of embryoid bodies (EB) culture to assess the effect of HDAC inhibition and proteasome pathway on cardiogenesis. HDAC inhibition stimulates the growth of EB, which is associated with faster spontaneous rhythmic contraction. HDAC inhibition increased the up-regulation of GATA4, MEF2, NKX2.5, cardiac actin, and α-SMA mRNA and protein levels that were abrogated by MG132. Immunostaining analysis demonstrates that trichostatin A and sodium butyrate resulted in a significant increase in cardiac lineage commitments that were blocked by the proteasome inhibition. Notably, HDAC inhibitors led to noticeable HDAC4 degradation, which was effectively prevented by MG132. Luciferase assay demonstrates an activation of MEF2 cardiac transcriptional factor by HDAC inhibition, which was repressed by MG132, revealing that the degradation of HDAC4 allows the activation of MEF2. Conclusions: Taken together, our study is the first to demonstrate that HDAC inhibition through proteasome pathway forms a novel signaling to determine the cardiac lineage commitment and elicit the survival pathway, which is dependent on specific HDAC4 degradation and subsequent MEF2 activation of ESCs.

scholarly journals A comprehensive review on application of stem cells for kidney diseases

2020 ◽  
Vol 11 (2) ◽  
pp. 1377-1385
Author(s):  
Chetan Kumar V H ◽  
Famna Roohi N K ◽  
Gowda D V
Keyword(s):  
Stem Cells ◽  
Bacterial Infections ◽  
Complex Disease ◽  
Ethical Issues ◽  
Kidney Diseases ◽  
Embryonic Stem ◽  
Turnaround Time ◽  
Embryoid Bodies ◽  
Renal Progenitor Cells ◽  
The Impact

The recognition of kidney failure as a complex disease requires multifactorial therapy in order to correct the conventional nonfactorial deficiency. Firstly, self-renewal means the ability of most organisms to reproduce without separation or aging; secondly, more than one form of a mature somatic cell is identified by each of the three regardless of kidney disorders, it can lead to loss of the environment, often bacterial infections. The reconstruction of the kidney has produced a spectacular response in this framework. The restoration of weakened and new kidneys is an alternative to renal replacement therapy. Both teratomas and embryoid bodies consist of three different layers of embryonic germs. Induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs) are present. These either provide useful therapeutical resources or can explore pathophysiology, including kidney diseases or infection. The benefit of ESCs is that they are relatively quick to receive and no longer subject to licensing/realty fees. Nevertheless, there are still some major concerns, such as ethical issues, the high risks to degeneration of neoplasm and immunocompatibility. The great benefit of iPSCs is that they have the same genetic history they drive making them an excellent method for studying the impact of genetic variants on disease path the key risk associated with the use of iPSCs are oogenesis, Tumorigenicity, and immunogenicity, the presence of an epigenetic memory, technical and economic issue associatedwith their long turnaround time and the presence of loyalties are the key risks associated with the use of iPSCs. Human pluripotent SCs have two major areas of use in kidney regeneration: they can be used by way organoid, scaffold,organ-on-a-chip, or blastocyst experiment to develop a "new kidney" or part of it. Renal progenitor cells are an alternative to either test or modulate regeneration of the kidney, offering significant benefits in the field. For encouraging us to hypothesize their medical use, a deeper understanding of the biology of pluripotent SCs is necessary.

scholarly journals Reduced chromatin binding of MYC is a key effect of HDAC inhibition in MYC amplified medulloblastoma

2020 ◽  
Author(s):  
Jonas Ecker ◽  
Venu Thatikonda ◽  
Gianluca Sigismondo ◽  
Florian Selt ◽  
Gintvile Valinciute ◽  
...  
Keyword(s):  
Molecular Interaction ◽  
Hdac Inhibitors ◽  
Class I ◽  
Chromatin Binding ◽  
Hdac Inhibition ◽  
Primary Tumors ◽  
Chip Sequencing ◽  
Class I Hdacs ◽  
The Impact ◽  
Selection Of

Abstract Background The sensitivity of myelocytomatosis oncogene (MYC) amplified medulloblastoma to class I histone deacetylase (HDAC) inhibition has been shown previously; however, understanding the underlying molecular mechanism is crucial for selection of effective HDAC inhibitors for clinical use. The aim of this study was to investigate the direct molecular interaction of MYC and class I HDAC2, and the impact of class I HDAC inhibition on MYC function. Methods Co-immunoprecipitation and mass spectrometry were used to determine the co-localization of MYC and HDAC2. Chromatin immunoprecipitation (ChIP) sequencing and gene expression profiling were used to analyze the co-localization of MYC and HDAC2 on DNA and the impact on transcriptional activity in primary tumors and a MYC amplified cell line treated with the class I HDAC inhibitor entinostat. The effect on MYC was investigated by quantitative real-time PCR, western blot, and immunofluorescence. Results HDAC2 is a cofactor of MYC in MYC amplified medulloblastoma. The MYC-HDAC2 complex is bound to genes defining the MYC-dependent transcriptional profile. Class I HDAC inhibition leads to stabilization and reduced DNA binding of MYC protein, inducing a downregulation of MYC activated genes (MAGs) and upregulation of MYC repressed genes (MRGs). MAGs and MRGs are characterized by opposing biological functions and by distinct enhancer-box distribution. Conclusions Our data elucidate the molecular interaction of MYC and HDAC2 and support a model in which inhibition of class I HDACs directly targets MYC’s transactivating and transrepressing functions.

scholarly journals Effect of ectopic expression of homeoprotein EGAM1C on the cell morphology, growth, and differentiation in a mouse embryonic stem cell line, MG1.19 cells

Reproduction ◽  
2012 ◽  
Vol 143 (4) ◽  
pp. 477-489 ◽  
Author(s):  
Momoe Iha ◽  
Masafumi Watanabe ◽  
Yumi Kihara ◽  
Saiko Sugawara ◽  
Koichi Saito ◽  
...  
Keyword(s):  
Es Cells ◽  
Expression System ◽  
Ectopic Expression ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies ◽  
Marker Genes ◽  
Undifferentiated State ◽  
The Impact

The homeoprotein EGAM1C was identified in preimplantation mouse embryos and embryonic stem (ES) cells. To explore the impact of EGAM1C on the hallmarks of mouse ES cells, MG1.19 cells stably expressing EGAM1C at levels similar to those in blastocysts were established using an episomal expression system. In the presence of leukemia inhibitory factor (+LIF), control transfectants with an empty vector formed flattened cell colonies, whileEgam1ctransfectants formed compacted colonies with increased E-CADHERIN expression. InEgam1ctransfectants, the cellular contents of POU5F1 (OCT4), SOX2, TBX3, and NANOG increased. Cell growth was accelerated in an undifferentiated state sustained by LIF and in the course of differentiation. During clonal proliferation, EGAM1C stabilized the undifferentiated state. In adherent culture conditions, EGAM1C partly inhibited the progression of differentiation at least within a 4-day culture period in the presence of retinoic acid by preventing the downregulation of LIF signaling with a robust increase in TBX3 expression. Conversely, EGAM1C enhanced the expression of lineage marker genesFgf5(epiblast),T(mesoderm),Gata6(primitive endoderm), andCdx2(trophectoderm) in −LIF conditions. In embryoid bodies expressing EGAM1C, the expression of marker genes for extraembryonic cell lineages, includingTpbpa(spongiotrophoblast) andPlat(parietal endoderm), increased. These results demonstrated that the ectopic expression of EGAM1C is capable of affecting the stabilization of an undifferentiated state and the progression of differentiation in MG1.19 ES cells, in addition to affecting cellular morphology and growth.

scholarly journals Biosimulation Using the Cellworks Computational Omics Biology Model (CBM) Identifies Genomic and Molecular Markers for Decitabine (DAC) Plus Valproic-Acid (VPA) Treatment Response in Patients with Myelodysplastic Syndromes (MDS)

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 2615-2615
Author(s):  
Michael Castro ◽  
Ansu Kumar ◽  
Himanshu Grover ◽  
Vivek Patil ◽  
Shweta Kapoor ◽  
...  
Keyword(s):  
Valproic Acid ◽  
Clinical Response ◽  
Copy Number ◽  
Disease Model ◽  
Hdac Inhibitors ◽  
Patient Specific ◽  
Beta Catenin ◽  
Hdac Inhibition ◽  
Specific Disease ◽  
The Impact

Abstract Background: DNA methyltransferase inhibition (DNMTi) with hypomethylating agents (HMA), azacitidine (AZA) or decitabine (DAC), remains the mainstay of therapy for most high-risk Myelodysplastic syndrome (MDS) patients. However, only 40-50% of MDS patients achieve clinical improvement with DNMTi. Previously, combinations of HMA and histone deacetylase (HDAC) inhibitors have been explored in MDS with varying clinical outcomes. However, the heterogeneity of genomic aberrations in MDS portend widely divergent responses from HDAC inhibition, implying that a predictive clinical decision support tool could select patients most likely to benefit from this combination. We explored the molecular basis of observed clinical response in a group of patients treated with DAC and Valproic-Acid (VPA). Method: 16 MDS patients with known clinical responses to DAC + VPA were selected for study from the Cellworks patient repository. The aberration and copy number variations from individual cases served as input into the Computational Omics Biology Model, a computational multi-omic biology software model largely created using literature sourced from PubMed, to generate a patient-specific protein network map. Disease biomarkers unique to each patient were identified within these maps. The Cellworks Biosimulation Platform has the capacity to biosimulate disease phenotypic behavior and was used to create a patient-specific disease model. Biosimulations were then conducted on each patient-specific disease model to measure the effect of DAC + VPA according to a cell growth score. This score was comprised of a composite of cell proliferation, viability, apoptosis, metastasis, and other cancer hallmarks. Biosimulation of drug response was conducted to identify and predict therapeutic efficacy. Results: In the biosimulation, VPA is a relatively weak HDAC inhibitor, but it also inhibits GSK3B and in turn increases beta-catenin (CTNNB1) levels. Additionally, monosomy 7 associated with loss of CAV1, HIPK2, and TRRAP also causes high CTNNB1, thereby further contributing to drug resistance. Biosimulation correctly identified that 7 of 8 patients with these genomic findings were clinical non-responders (NR) to VPA, indicating that CTNNB1 status is likely to predict treatment failure from the VPA + HMA combination in this disease. Notably, CTNNB1 levels have been reported to foster an immune-evasive tumor microenvironment resistant to CTL activation. By contrast, high levels of c-MYC predict response to VPA + HMA combination. VPA inhibits MYC transcription and thereby reduces MYC-induced downregulation of p21 through CKS1B. Additionally MYC is a transcriptional regulator of DNMT1 which is degraded after hyperacetylation induced by HDAC3 inhibition suggesting that VPA also enhances DNMT1 turnover. One patient analyzed had trisomy 8 resulting in c-MYC over-expression and responded to HMA + VPA. Additionally, other aberrations enhancing c-MYC transcription such as copy number variant (CNV) loss of MXI1, HHEX, FBXW7, SMAD7 or CNV gain of BRD4, BCL7B led to high clinical response to the combination (Table 1). By comparison to the CTNNB1-driven subset, the impact of VPA on CTNNB1 in the MYC-dominant disease network did not negate the benefit of VPA for these patients. Additionally, the inhibition of GSK3B by VPA leading to diminished FBXW7 and less ubiquitin-mediated turnover of c-MYC was not sufficient to overcome the inhibition of MYC transcription and HDAC3i-mediated turnover. Immune activation has become a recognized mechanism of responsiveness to HMA. However, among patients with upregulated CTNNB1, VPA is likely to further decrease response to treatment. By contrast, among MYC-driven cancers that are typically immune-evasive, VPA appears to be a vital mechanism of overcoming MYC-driven immune evasion. Conclusion: Signaling pathway consequences related to CTNNB1 and c-MYC upregulation predict response to DAC + VPA. Although HMA plus HDAC inhibition can be generally beneficial for MDS, variable mechanisms of action among various HDAC inhibitors and unique patient disease characteristics should be considered for optimal treatment selection. Finally, CTNNB1 emerged from the Cellworks biosimulations as a therapeutically relevant target in MDS that determines whether VPA synergizes or antagonizes the effect of other agents in this challenging subtype of MDS. Figure 1 Figure 1. Disclosures Castro: Caris Life Sciences Inc.: Consultancy; Omicure Inc: Consultancy; Cellworks Group Inc.: Current Employment; Exact sciences Inc.: Consultancy; Guardant Health Inc.: Speakers Bureau; Bugworks: Consultancy. Kumar: Cellworks Group Inc.: Current Employment. Grover: Cellworks Group Inc.: Current Employment. Patil: Cellworks Group Inc.: Current Employment. Kapoor: Cellworks Group Inc.: Current Employment. Agrawal: Cellworks Group Inc.: Current Employment. Sauban: Cellworks Group Inc.: Current Employment. Prasad: Cellworks Group Inc.: Current Employment. Basu: Cellworks Group Inc.: Current Employment. Suseela: Cellworks Group Inc.: Current Employment. Kumar: Cellworks Group Inc.: Current Employment. Nair: Cellworks Group Inc.: Current Employment. Kumari: Cellworks Group Inc.: Current Employment. Pampana: Cellworks Group Inc.: Current Employment. Ullal: Cellworks Group Inc.: Current Employment. Azam: Cellworks Group Inc.: Current Employment. Prasad: Cellworks Group Inc.: Current Employment. Amara: Cellworks Group Inc.: Current Employment. Sahu: Cellworks Group Inc.: Current Employment. Raveendaran: Cellworks Group Inc.: Current Employment. Veedu: Cellworks Group Inc.: Current Employment. Mundkur: Cellworks Group Inc: Current Employment. Patel: Cellworks Group Inc.: Current Employment. Christie: Cellworks Group Inc.: Current Employment. Macpherson: Cellworks Group Inc.: Current Employment. Howard: Servier: Consultancy; Cellworks Group Inc.: Consultancy; Sanofi: Consultancy, Other: Speaker fees.

Histone Deacetylase Inhibitors Enhance Cell Death In Burkitt Lymphoma Cells By Modulating Expression Of The WNT/β-Catenin Pathway and Survivin

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1291-1291
Author(s):  
Faye Pais ◽  
Matthew J. Barth ◽  
Rodney R. Miles ◽  
Michelle L. Hermiston
Keyword(s):  
Cell Death ◽  
Western Blot ◽  
Histone Deacetylase ◽  
Burkitt Lymphoma ◽  
Caspase 3 ◽  
Hdac Inhibitors ◽  
Vehicle Control ◽  
Hdac Inhibition ◽  
Raji Cells ◽  
Apoptotic Proteins

Abstract Background Burkitt Lymphoma (BL) is an aggressive non-Hodgkin B cell neoplasm primarily affecting children. While overall cure rates are high, prognosis for the 20% of patients with relapsed or refractory is dismal with a 16% survival rate at four years, indicating the need for novel therapeutic approaches. While epigenetic modulators have shown potential therapeutic benefit in other hematologic malignancies, their use in BL has been limited. Here, we evaluate whether histone deacetylase (HDAC) inhibitors could enhance cell death in chemotherapy resistant BL cells. Methods The BL cell lines Ramos, Raji, or previously generated/characterized immunochemotherapy resistant Raji BL cell lines (Raji 2R and Raji 4RH) (Barth et al., Br J Haematol, 2012) were treated for 48 hours with 4-hydroperoxycyclophosphamide (4-HC, the active metabolite of cyclophosphamide), cytarabine, prednisolone, HDAC inhibitors (suberoyanilide hydroxamic acid (SAHA, vorinostat), and romidepsin), or vehicle control. Cell viability was measured on a Via Cell, by Alamar Blue staining (Invitrogen), or by measuring Caspase-3 activation by flow cytometry. Levels of pro- (survivin, XIAP, MCL-1, livin, and BCL-XL) and anti- (Bax, Bak) apoptotic proteins were evaluated using standard western blot techniques. To evaluate the wiring of signaling networks in the basal state or in response to drug treatment, samples were fixed, permeabilized, and simultaneously stained for Caspase-3, phospho GSK-3 (pGSK3, the inactive form of this protein), active β-catenin, and survivin. Cells were processed on a BD FACSVerse and analyzed by gating on Caspase-3 negative (chemotherapy resistant) cells and monitoring the activity of the pGSK3, active β-catenin, and survivin using FlowJO (V9.6) software. Results Relative to the BL cell line Ramos, Raji BL cells were resistant to all doses of conventional chemotherapy (cyclophosphamide, ara-c, and prednisolone) tested. Because hyper-activation of the WNT/b-catenin network due to epigenetic modulation has been implicated in chemotherapy resistance in other B-lineage pediatric malignancies (Hogan et al., Blood 2011), we treated cells with chemotherapy or vehicle and used phosphoflow cytometry to evaluate the expression of pGSK-3, active β-catenin, and its downstream target survivin. Relative to vehicle control, we found that exposure to cytotoxic chemotherapy resulted in rewiring of the cellular networks with increased levels of pGSK3, β-catenin, and survivin. Western blot analyses of the immunochemotherapy resistant Raji 2R and Raji 4RH cells revealed similar upregulation of anti-apoptotic proteins including survivin, downregulation of pro-apoptotic proteins Bak and Bax, and increased expression of pGSK3 compared to Raji cells. To test whether alterations in these signaling axes might enhance the survival of chemorefractory BL cells, we treated Raji cells with the HDAC inhibitor SAHA and found induction of apoptosis at nanomolar doses. Sensitivity to SAHA correlated with down-regulation of active β-catenin and survivin in a dose-dependent manner. To extend these results, we also tested whether immunochemotherapy resistant Raji 2R and Raji 4RH cells were sensitive to HDAC inhibition and found similar robust induction of cell death upon treatment with a panel of HDAC inhibitors. Conclusions Active β-catenin and survivin are upregulated in chemotherapy resistant BL cells. HDAC inhibition results in their rapid down regulation and enhanced apoptotic cell death. Further evaluation of inhibitors of the WNT/ β-catenin pathway and HDAC inhibitors is warranted in chemotherapy resistant BL and could have a role in treating refractory or relapsed BL patients. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Regulation of Programmed Cell Death by Basement Membranes in Embryonic Development

2000 ◽  
Vol 150 (5) ◽  
pp. 1215-1221 ◽  
Author(s):  
Patricia Murray ◽  
David Edgar
Keyword(s):  
Cell Death ◽  
Programmed Cell Death ◽  
Embryonic Stem ◽  
Basement Membranes ◽  
Embryoid Bodies ◽  
Wild Type ◽  
Mammalian Embryo ◽  
Epithelial Development ◽  
Endodermal Cells

The formation of the proamniotic cavity in the mammalian embryo is the earliest of many instances throughout development in which programmed cell death and the formation of epithelia play fundamental roles (Coucouvanis, E., and G.R. Martin. 1995. Cell. 83:279–287). To determine the role of the basement membrane (BM) in cavitation, we use embryoid bodies derived from mouse embryonic stem cells in which the LAMC1 genes have been inactivated to prevent BM deposition (Smyth, N., H.S. Vatansever, P. Murray, M. Meyer, C. Frie, M. Paulsson, and D. Edgar. 1999. J. Cell Biol. 144:151–610). We demonstrate here that LAMC1−/− embryoid bodies are unable to cavitate, and do not form an epiblast epithelium in the absence of a BM, although both embryonic ectodermal cells and extraembryonic endodermal cells do differentiate, as evidenced by the expression of cell-specific markers. Acceleration or rescue of BM deposition by exogenous laminin in wild-type or LAMC1−/− embryoid bodies, respectively, results in cavitation that is temporally and spatially associated with restoration of epiblast epithelial development. We conclude that the BM not only directly regulates development of epiblast epithelial cells, but also indirectly regulates the programmed cell death necessary for cavity formation.

Novel Conjugated Quinazolinone-Based Hydroxamic Acids: Design, Synthesis and Biological Evaluation

2020 ◽  
Vol 16 ◽  
Author(s):  
Tran Khac Vu ◽  
Nguyen Thi Thanh ◽  
Nguyen Van Minh ◽  
Nguyen Huong Linh ◽  
Nguyen Thi Phương Thao ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Cell Lines ◽  
Cancer Cell ◽  
Hdac Inhibitors ◽  
Cancer Cell Lines ◽  
Hydroxamic Acids ◽  
Biological Evaluation ◽  
Hdac Inhibition ◽  
Ic50 Value ◽  
Mcf 7

Background: Target-based approach to drug discovery currently attracts a great deal of interest from medicinal chemists in anticancer drug discovery and development. Histone deacetylase (HDAC) inhibitors represent an extensive class of targeted anti-cancer agents. Among the most explored structure moieties, hydroxybenzamides and hydroxypropenamides have been demonstrated to have potential HDAC inhibitory effects. Several compounds of these structural classes have been approved for clinical uses to treat different types of cancer, such as vorinostat and belinostat. Aims: This study aims at developing novel HDAC inhibitors bearing conjugated quinazolinone scaffolds with potential cytotoxicity against different cancer cell lines. Method: A series of novel N-hydroxyheptanamides incorporating conjugated 6-hydroxy-2 methylquinazolin-4(3H)- ones (15a-l) was designed, synthesized and evaluated for HDAC inhibitory potency as well as cytotoxicity against three human cancer cell lines, including HepG-2, MCF-7 and SKLu-1. Molecular simulations were finally performed to gain more insight into the structure-activity. relationships. Results: It was found that among novel conjugated quinazolinone-based hydroxamic acids synthesized, compounds 15a, 15c and 15f were the most potent, both in terms of HDAC inhibition and cytotoxicity. Especially, compound 15f displayed up to nearly 4-fold more potent than SAHA (vorinostat) in terms of cytotoxicity against MCF-7 cell line with IC50 value of 1.86 µM, and HDAC inhibition with IC50 value of 6.36 µM. Docking experiments on HDAC2 isozyme showed that these compounds bound to HDAC2 with binding affinities ranging from -10.08 to -14.93 kcal/mol compared to SAHA (-15.84 kcal/mol). It was also found in this research that most of the target compounds seemed to be more cytotoxic toward SKLu-1than MCF-7 and HepG-2. Conclusion: The resesrch results suggest that some hydroxamic acids could emerge for further evaluation and the results are well served as basics for further design of more potent HDAC inhibitors and antitumor agents.

scholarly journals Creating a human-induced pluripotent stem cell-based NKX2.5 reporter gene assay for developmental toxicity testing

2021 ◽  
Author(s):  
Karin Lauschke ◽  
Andreas Frederik Treschow ◽  
Mikkel Aabech Rasmussen ◽  
Nichlas Davidsen ◽  
Bjørn Holst ◽  
...  
Keyword(s):  
Stem Cell ◽  
Reporter Gene ◽  
Luminescence Intensity ◽  
Developmental Toxicity ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Luciferase Reporter ◽  
Reporter Cell ◽  
Gene Assay ◽  
Induced Pluripotent

AbstractTo test large numbers of chemicals for developmental toxicity, rapid in vitro tests with standardized readouts for automated data acquisition are needed. However, the most widely used assay, the embryonic stem cell test, relies on the counting of beating embryoid bodies by visual inspection, which is laborious and time consuming. We previously developed the PluriBeat assay based on differentiation of human induced pluripotent stem cells (hiPSC) that we demonstrated to be predictive for known teratogens at relevant concentrations using the readout of beating cardiomyocytes. Here, we report the development of a novel assay, which we term the PluriLum assay, where we have introduced a luciferase reporter gene into the locus ofNKX2.5of our hiPSC line. This enabled us to measure luminescence intensities instead of counting beating cardiomyocytes, which is less labor intensive. We established twoNKX2.5reporter cell lines and validated their pluripotency and genetic stability. Moreover, we confirmed that the genetically engineeredNKX2.5reporter cell line differentiated into cardiomyocytes with the same efficiency as the original wild-type line. We then exposed the cells to valproic acid (25–300 μM) and thalidomide (0.1–36 µM) and compared the PluriBeat readout of the cardiomyocytes with the luminescence intensity of the PluriLum assay. The results showed that thalidomide decreased luminescence intensity significantly with a higher potency and efficacy compared to the beating readout. With this, we have developed a novel hiPSC-based assay with a standardized readout that may have the potential for higher throughput screening for developmental toxicity.

scholarly journals Modulation of Differentiation of Embryonic Stem Cells by Polypyrrole: The Impact on Neurogenesis

2021 ◽  
Vol 22 (2) ◽  
pp. 501
Author(s):  
Kateřina Skopalová ◽  
Katarzyna Anna Radaszkiewicz ◽  
Věra Kašpárková ◽  
Jaroslav Stejskal ◽  
Patrycja Bober ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Stem Cell Differentiation ◽  
Active Role ◽  
Low Molecular Weight ◽  
Conducting Materials ◽  
The Impact ◽  
Erk Kinase

The active role of biomaterials in the regeneration of tissues and their ability to modulate the behavior of stem cells in terms of their differentiation is highly advantageous. Here, polypyrrole, as a representantive of electro-conducting materials, is found to modulate the behavior of embryonic stem cells. Concretely, the aqueous extracts of polypyrrole induce neurogenesis within embryonic bodies formed from embryonic stem cells. This finding ledto an effort to determine the physiological cascade which is responsible for this effect. The polypyrrole modulates signaling pathways of Akt and ERK kinase through their phosphorylation. These effects are related to the presence of low-molecular-weight compounds present in aqueous polypyrrole extracts, determined by mass spectroscopy. The results show that consequences related to the modulation of stem cell differentiation must also be taken into account when polypyrrole is considered as a biomaterial.

scholarly journals miR-380-5p facilitates NRF2 and attenuates cerebral ischemia/reperfusion injury-induced neuronal cell death by directly targeting BACH1

2021 ◽  
Vol 12 (1) ◽  
pp. 210-217
Author(s):  
Yibiao Wang ◽  
Min Xu
Keyword(s):  
Cell Death ◽  
Cerebral Ischemia ◽  
Reperfusion Injury ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
Neuronal Cell Death ◽  
Neuronal Cell ◽  
Luciferase Assay ◽  
Neuroprotective Effects ◽  
Cerebral Ischemia Reperfusion

Abstract Background This study aimed to explore the role of miR-380-5p in cerebral ischemia/reperfusion (CIR) injury-induced neuronal cell death and the potential signaling pathway involved. Methodology Human neuroblastoma cell line SH-SY5Y cells were used in this study. Oxygen and glucose deprivation/reperfusion (OGD/R) model was used to mimic ischemia/reperfusion injury. CCK-8 assay and flow cytometry were used to examine cell survival. Quantitative real time PCR (RT-qPCR) assay and Western blotting were used to measure the change of RNA and protein expression, respectively. TargetScan and Luciferase assay was used to confirm the target of miR-380-5p. Malondialdehyde (MDA) superoxide dismutase (SOD) and glutathione peroxidase (GSHPx) were measured using commercial kits. Results miR-380-5p was downregulated in SH-SY5Y cells after OGD/R. Cell viability was increased by miR-380-5p, while cell apoptosis was reduced by miR-380-5p mimics. MDA was reduced by miR-380-5p mimics, while SOD and GSHPx were increased by miR-380-5p. Results of TargetScan and luciferase assay have showed that BACH1 is the direct target of miR-380-5p. Expression of NRF2 was upregulated after OGD/R, but was not affected by miR-380-5p. mRNA expression of HO-1 and NQO1 and ARE activity were increased by miR-380-5p. Overexpression of BACH1 reversed the antioxidant and neuroprotective effects of miR-380-5p. Conclusion miR-380-5p inhibited cell death induced by CIR injury through target BACH1 which also facilitated the activation of NRF2, indicating the antioxidant and neuroprotective effects of miR-380-5p.

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