203 ESTABLISHMENT OF AN IN VITRO SCREENING ASSAY FOR DEVELOPMENTAL TOXICITY USING MOUSE EMBRYOID BODIES

2014 ◽  
Vol 26 (1) ◽  
pp. 216
Author(s):  
Y. Choi ◽  
E. B. Jeung
Keyword(s):  
Developmental Toxicity ◽  
Marker Gene ◽  
Embryoid Bodies ◽  
Toxic Chemicals ◽  
Penicillin G ◽  
Marker Genes ◽  
High Dose ◽  
Germ Layers ◽  
Hes Cells

Toxicity is generally referred to as the degree to which a substance can damage an organism. According to numerous reports, determination of toxicity through an in vitro system is an economical and ethical method, compared with an in vivo system using animals. The hES cells can differentiate into three germ layers and the unique feature of hES cells from the three germ layers via the EB formation adds good insight in understanding human developmental biology in vitro. Evaluation of developmental toxicity of a compound on early embryo states can therefore be based on its effect on EB formation. In this study, we used mouse embryonic stem cells (mESC) to investigate the effects of several developmental toxic chemicals on the formation of EBs. We used the EB hanging drop method and tested five toxic chemicals; cytosine arabinoside, dexamethasone, hydroxyurea, indomethacin, 5-fluorouracil, and two negative controls; ascorbic acid and penicillin G. We demonstrated a significant reduction of EB size after treatment with a high dose of each chemical. We evaluated cell toxicity by performing measurements of cell viability after treatment with each chemical. Expression of apoptosis-related genes (p53, Sirt1, p21, Puma, Noxa, Mdm2) and pluripotency marker genes (Oct4, Nanog, Sox2, ZFP206) and differentiation marker genes, such as endoderm (HNF4, AFP), mesoderm (T-brachy), and ectoderm (Pax6) were determined by quantitative real-time PCR. The chemicals induced abnormal differentiation and apoptosis. We confirmed apoptotic positive cells by TUNEL assay. In addition, we also demonstrate the reduction of the size of EBs through expression of apoptosis-related marker genes (p53, Caspase-3, PARP) and necrosis-related marker gene (HMGB1). The results obtained demonstrate that EBs can be used as an in vitro model for testing developmental toxicity.

scholarly journals Insertion of EGFP into the replicase gene of Semliki Forest virus results in a novel, genetically stable marker virus

2007 ◽  
Vol 88 (4) ◽  
pp. 1225-1230 ◽  
Author(s):  
Nele Tamberg ◽  
Valeria Lulla ◽  
Rennos Fragkoudis ◽  
Aleksei Lulla ◽  
John K. Fazakerley ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Semliki Forest Virus ◽  
Cultured Cells ◽  
Marker Gene ◽  
Virus Production ◽  
Marker Genes ◽  
Replicase Gene ◽  
Marker Virus

Alphavirus-based vector and replicon systems have been extensively used experimentally and are likely to be used in human and animal medicine. Whilst marker genes can be inserted easily under the control of a duplicated subgenomic promoter, these constructs are often genetically unstable. Here, a novel alphavirus construct is described in which an enhanced green fluorescent protein (EGFP) marker gene is inserted into the virus replicase open reading frame between nsP3 and nsP4, flanked by nsP2 protease-recognition sites. This construct has correct processing of the replicase polyprotein, produces viable virus and expresses detectable EGFP fluorescence upon infection of cultured cells and cells of the mouse brain. In comparison to parental virus, the marker virus has an approximately 1 h delay in virus RNA and infectious virus production. Passage of the marker virus in vitro and in vivo demonstrates good genetic stability. Insertion of different markers into this novel construct has potential for various applications.

scholarly journals Pilot investigation on the dose-dependent impact of irradiation on primary human alveolar osteoblasts in vitro

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Anna-Klara Amler ◽  
Domenic Schlauch ◽  
Selin Tüzüner ◽  
Alexander Thomas ◽  
Norbert Neckel ◽  
...  
Keyword(s):  
Bone Cells ◽  
Marker Gene ◽  
Marker Genes ◽  
Dependent Manner ◽  
Current Standard ◽  
Functional Changes ◽  
Standard Procedures ◽  
The Impact ◽  
Dose Dependent

AbstractRadiotherapy of head and neck squamous cell carcinoma can lead to long-term complications like osteoradionecrosis, resulting in severe impairment of the jawbone. Current standard procedures require a 6-month wait after irradiation before dental reconstruction can begin. A comprehensive characterization of the irradiation-induced molecular and functional changes in bone cells could allow the development of novel strategies for an earlier successful dental reconstruction in patients treated by radiotherapy. The impact of ionizing radiation on the bone-forming alveolar osteoblasts remains however elusive, as previous studies have relied on animal-based models and fetal or animal-derived cell lines. This study presents the first in vitro data obtained from primary human alveolar osteoblasts. Primary human alveolar osteoblasts were isolated from healthy donors and expanded. After X-ray irradiation with 2, 6 and 10 Gy, cells were cultivated under osteogenic conditions and analyzed regarding their proliferation, mineralization, and expression of marker genes and proteins. Proliferation of osteoblasts decreased in a dose-dependent manner. While cells recovered from irradiation with 2 Gy, application of 6 and 10 Gy doses not only led to a permanent impairment of proliferation, but also resulted in altered cell morphology and a disturbed structure of the extracellular matrix as demonstrated by immunostaining of collagen I and fibronectin. Following irradiation with any of the examined doses, a decrease of marker gene expression levels was observed for most of the investigated genes, revealing interindividual differences. Primary human alveolar osteoblasts presented a considerably changed phenotype after irradiation, depending on the dose administered. Mechanisms for these findings need to be further investigated. This could facilitate improved patient care by re-evaluating current standard procedures and investigating faster and safer reconstruction concepts, thus improving quality of life and social integrity.

scholarly journals Directing Stem Cell Commitment by Amorphous Calcium Phosphate Nanoparticles Incorporated in PLGA: Relevance of the Free Calcium Ion Concentration

2020 ◽  
Vol 21 (7) ◽  
pp. 2627
Author(s):  
Olivier Gröninger ◽  
Samuel Hess ◽  
Dirk Mohn ◽  
Elia Schneider ◽  
Wendelin Stark ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Calcium Phosphate ◽  
Amorphous Calcium Phosphate ◽  
Marker Gene ◽  
Ion Concentration ◽  
Marker Genes ◽  
Calcium Phosphate Nanoparticles ◽  
Cell Commitment

The microenvironment of mesenchymal stem cells (MSCs) is responsible for the modulation in MSC commitment. Nanocomposites with an inorganic and an organic component have been investigated, and osteogenesis of MSCs has been attributed to inorganic phases such as calcium phosphate under several conditions. Here, electrospun meshes and two-dimensional films of poly(lactic-co-glycolic acid) (PLGA) or nanocomposites of PLGA and amorphous calcium phosphate nanoparticles (PLGA/aCaP) seeded with human adipose-derived stem cells (ASCs) were analyzed for the expression of selected marker genes. In a two-week in vitro experiment, osteogenic commitment was not found to be favored on PLGA/aCaP compared to pure PLGA. Analysis of the medium revealed a significant reduction of the Ca2+ concentration when incubated with PLGA/aCaP, caused by chemical precipitation of hydroxyapatite (HAp) on aCaP seeds of PLGA/aCaP. Upon offering a constant Ca2+ concentration, however, the previously observed anti-osteogenic effect was reversed: alkaline phosphatase, an early osteogenic marker gene, was upregulated on PLGA/aCaP compared to pristine PLGA. Hence, in addition to the cell–material interaction, the material–medium interaction was also important for the stem cell commitment here, affecting the cell–medium interaction. Complex in vitro models should therefore consider all factors, as coupled impacts might emerge.

182 Establishment of expanded potential embryonic stem cell lines from porcine embryos

2019 ◽  
Vol 31 (1) ◽  
pp. 215
Author(s):  
M. Nowak-Imialek ◽  
X. Gao ◽  
P. Liu ◽  
H. Niemann
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Cell Lines ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Differentiation Potential ◽  
Germ Layers ◽  
Embryonic Tissues

The domestic pig is an excellent large animal in biomedical medicine and holds great potential for testing the clinical safety and efficacy of stem cell therapies. Previously, numerous studies reported the derivation of porcine embryonic stem cell (ESC)-like lines, but none of these lines fulfilled the stringent criteria for true pluripotent germline competent ESC. Here, we report the first establishment of porcine expanded potential stem cells (pEPSC) from parthenogenetic and in vivo-derived blastocysts. A total of 12 cell lines from parthenogenetic blastocysts from Day 7 (12/24) and 26 cell lines from in vivo-derived blastocysts from Day 5 (26/27) were established using defined stem cell culture conditions. These cells closely resembled mouse ESC with regard to morphology, formed compact colonies with high nuclear/cytoplasmic ratios, and could be maintained in vitro for more than 40 passages with a normal karyotype. The pEPSC expressed key pluripotency genes, including OCT4, NANOG, SOX2, and SALL4 at similar levels as porcine blastocysts. Immunostaining analysis confirmed expression of critical cell surface markers SSEA-1 and SSEA-4 in pEPSC. The EPSC differentiated in vitro into tissues expressing markers of the 3 germ layers: SOX7, AFP, T, DES, CRABP2, α-SMA, β-tubulin, PAX6, and, notably, the trophoblast markers HAND1, GATA3, PGF, and KRT7. After injection into immunocompromised mice, the pEPSC formed teratomas with derivatives of the 3 germ layers and placental lactogen-1 (PL-1)-positive trophoblast-like cells. Additionally, pEPSC cultured in vitro under conditions specific for germ cells formed embryoid bodies, which contained ~9% primordial germ cell (PGC)-like cells (PGCLC) that expressed PGC-specific genes, including NANOS3, BLIMP1, TFAP2C, CD38, DND1, KIT, and OCT4 as detected by quantitative RT-PCR and immunostaining. Next, we examined the in vivo differentiation potential of pEPSC and injected pEPSC stably expressing the CAG-H2B-mCherry transgene reporter into porcine embryos. The donor cells proliferated and were localised in both the trophectoderm and inner cell mass of the blastocysts cultured in vitro. After transfer to 3 recipient sows, chimeric embryos implanted and a total of 45 fetuses were recovered on Days 26 to 28. Flow cytometry of single cells collected from embryonic and extraembryonic tissues of the fetuses revealed mCherry+ cells in 7 conceptuses, in both the placenta and embryonic tissues; in 3 chimeric conceptuses, mCherry+ cells were exclusively found in embryonic tissues; and in 2 conceptuses, mCherry+ cells were exclusively localised in the placenta. The contribution of the mCherry+ cells was low (0.4-1.7%), but they were found and co-detected in multiple porcine embryonic tissues using tissue lineage-specific markers, including SOX2, TUJ1, GATA4, SOX17, AFP, α-SMA, and trophoblast markers PL-1 and KRT7 in the placental cells. The successful establishment of pEPSC represents a major step forward in stem cell research and provides cell lines with the unique state of cellular potency useful for genetic engineering and unravelling pluripotency regulation in pigs.

scholarly journals A plant regeneration platform to apply new breeding techniques for improving disease resistance in grapevine rootstocks and cultivars

2019 ◽  
Vol 12 ◽  
pp. 01019 ◽  
Author(s):  
S. Sabbadini ◽  
L. Capriotti ◽  
C. Limera ◽  
O. Navacchi ◽  
G. Tempesta ◽  
...  
Keyword(s):  
Marker Gene ◽  
Marker Genes ◽  
Wine Grape ◽  
Thompson Seedless ◽  
Multiple Virus ◽  
Almost All ◽  
New Breeding Techniques ◽  
Breeding Techniques ◽  
Selection Of

Worldwide grapevine cultivation is based on the use of elite cultivars, in many cases strictly linked to local important wine brands. Most of Vitis viniferacultivars have high susceptibility to fungal and viral diseases therefore, new breeding techniques (e.g. Cisgenesis, RNAi and gene editing) offer the possibility to introduce new clones of the main cultivars with increased diseases resistance, in order to reduce environmental impact and improve quality in the intensive wine grape industry. This study is finalized to develop efficient in vitro regeneration and transformation protocols to extend the application of these technologies in wine grape cultivars and rootstocks. With this aim, in vitro regeneration protocols based on the production of meristematic bulks (Mezzetti et al., 2002) were optimized for different grapevine cultivars (Glera, Vermentino, Sangiovese, Thompson Seedless) and rootstocks (1103 Paulsen, and 110 Richter). The meristematic bulks were then used as explants for Agrobacteriummediated genetic transformation protocols, by comparing the use of NPTII and e-GFP as marker genes. Results confirmed the efficiency of meristematic bulks as the regenerating tissue to produce new modified plants in almost all the above genotypes. The highest regeneration efficiency in some genotypes allowed the selection of stable modified lines/calli with only the use of e-GFP marker gene. This protocol can be applied in the use of MYB marker gene for the production of cisgenic lines. Genotypes having the highest regeneration and transformation efficiency were also used for transformation experiments using a hairpin gene construct designed to silence the RNA-dependent RNA polymerase (RpRd) of the GFLV and GLRaV3, which would induce multiple virus resistances, and the Dicer-like protein 1 (Bc-DCL1) and Bc-DCL2 to control B. cinerea infection.

Transforming growth factor-β1 signaling contributes to development of smooth muscle cells from embryonic stem cells

2004 ◽  
Vol 287 (6) ◽  
pp. C1560-C1568 ◽  
Author(s):  
Sanjay Sinha ◽  
Mark H. Hoofnagle ◽  
Paul A. Kingston ◽  
Mary E. McCanna ◽  
Gary K. Owens
Keyword(s):  
Smooth Muscle ◽  
Growth Factor ◽  
Transforming Growth Factor ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Model System ◽  
Embryoid Bodies ◽  
Regulatory Pathways ◽  
Tgf Β1

Knockout of transforming growth factor (TGF)-β1 or components of its signaling pathway leads to embryonic death in mice due to impaired yolk sac vascular development before significant smooth muscle cell (SMC) maturation occurs. Thus the role of TGF-β1 in SMC development remains unclear. Embryonic stem cell (ESC)-derived embryoid bodies (EBs) recapitulate many of the events of early embryonic development and represent a more physiological context in which to study SMC development than most other in vitro systems. The present studies showed induction of the SMC-selective genes smooth muscle α-actin (SMαA), SM22α, myocardin, smoothelin-B, and smooth muscle myosin heavy chain (SMMHC) within a mouse ESC-EB model system. Significantly, SM2, the SMMHC isoform associated with fully differentiated SMCs, was expressed. Importantly, the results showed that aggregates of SMMHC-expressing cells exhibited visible contractile activity, suggesting that all regulatory pathways essential for development of contractile SMCs were functional in this in vitro model system. Inhibition of endogenous TGF-β with an adenovirus expressing a soluble truncated TGF-β type II receptor attenuated the increase in SMC-selective gene expression in the ESC-EBs, as did an antibody specific for TGF-β1. Of interest, the results of small interfering (si)RNA experiments provided evidence for differential TGF-β-Smad signaling for an early vs. late SMC marker gene in that SMαA promoter activity was dependent on both Smad2 and Smad3 whereas SMMHC activity was Smad2 dependent. These results are the first to provide direct evidence that TGF-β1 signaling through Smad2 and Smad3 plays an important role in the development of SMCs from totipotential ESCs.

In vitro culture of stem-like cells derived from somatic cell nuclear transfer bovine embryos of the Korean beef cattle species, HanWoo

2016 ◽  
Vol 28 (11) ◽  
pp. 1762 ◽  
Author(s):  
Daehwan Kim ◽  
Sangkyu Park ◽  
Yeon-Gil Jung ◽  
Sangho Roh
Keyword(s):  
Beef Cattle ◽  
Somatic Cell ◽  
Somatic Cell Nuclear Transfer ◽  
Nuclear Transfer ◽  
Embryoid Bodies ◽  
Marker Genes ◽  
Bovine Embryos ◽  
In Vivo Differentiation

We established and maintained somatic cell nuclear transfer embryo-derived stem-like cells (SCNT-eSLCs) from the traditional Korean beef cattle species, HanWoo (Bos taurus coreanae). Each SCNT blastocyst was placed individually on a feeder layer with culture medium containing three inhibitors of differentiation (3i). Primary colonies formed after 2–3 days of culture and the intact colonies were passaged every 5–6 days. The cells in each colony showed embryonic stem cell-like morphologies with a distinct boundary and were positive to alkaline phosphatase staining. Immunofluorescence and reverse transcription–polymerase chain reaction analyses also confirmed that these colonies expressed pluripotent markers. The colonies were maintained over 50 passages for more than 270 days. The cells showed normal karyotypes consisting of 60 chromosomes at Passage 50. Embryoid bodies were formed by suspension culture to analyse in vitro differentiation capability. Marker genes representing the differentiation into three germ layers were expressed. Typical embryonal carcinoma was generated after injecting cells under the testis capsule of nude mice, suggesting that the cultured cells may also have the potential of in vivo differentiation. In conclusion, we generated eSLCs from SCNT bovine embryos, using a 3i system that sustained stemness, normal karyotype and pluripotency, which was confirmed by in vitro and in vivo differentiation.

scholarly journals A novel human pluripotent stem cell-based assay to predict developmental toxicity

2020 ◽  
Vol 94 (11) ◽  
pp. 3831-3846
Author(s):  
Karin Lauschke ◽  
Anna Kjerstine Rosenmai ◽  
Ina Meiser ◽  
Julia Christiane Neubauer ◽  
Katharina Schmidt ◽  
...  
Keyword(s):  
Developmental Toxicity ◽  
Three Dimensional ◽  
Cell Types ◽  
Microtiter Plate ◽  
Embryoid Bodies ◽  
The Body ◽  
Chemically Induced ◽  
Novel Method ◽  
Induced Pluripotent

Abstract There is a great need for novel in vitro methods to predict human developmental toxicity to comply with the 3R principles and to improve human safety. Human-induced pluripotent stem cells (hiPSC) are ideal for the development of such methods, because they are easy to retrieve by conversion of adult somatic cells and can differentiate into most cell types of the body. Advanced three-dimensional (3D) cultures of these cells, so-called embryoid bodies (EBs), moreover mimic the early developing embryo. We took advantage of this to develop a novel human toxicity assay to predict chemically induced developmental toxicity, which we termed the PluriBeat assay. We employed three different hiPSC lines from male and female donors and a robust microtiter plate-based method to produce EBs. We differentiated the cells into cardiomyocytes and introduced a scoring system for a quantitative readout of the assay—cardiomyocyte contractions in the EBs observed on day 7. Finally, we tested the three compounds thalidomide (2.3–36 µM), valproic acid (25–300 µM), and epoxiconazole (1.3–20 µM) on beating and size of the EBs. We were able to detect the human-specific teratogenicity of thalidomide and found the rodent toxicant epoxiconazole as more potent than thalidomide in our assay. We conclude that the PluriBeat assay is a novel method for predicting chemicals’ adverse effects on embryonic development.

scholarly journals Development of marker-free transgenic pigeon pea (Cajanus cajan) expressing a pod borer insecticidal protein

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Snehasish Sarkar ◽  
Souri Roy ◽  
Sudip K. Ghosh
Keyword(s):  
Insect Pests ◽  
Marker Gene ◽  
Pigeon Pea ◽  
Small Subunit ◽  
Grain Legume ◽  
Marker Genes ◽  
35S Promoter ◽  
Selectable Marker Genes ◽  
Insect Resistant

AbstractPigeon pea, a grain legume of the semiarid tropics, is a rich source of high-quality protein. The productivity of this pulse is seriously affected by lepidopteron insect pests. To generate a sustainable insect-resistant plant, synthetically prepared bioactive key constituents of a crystal protein (Syn Cry1Ab) of Bacillus thuringiensis were expressed in pigeon pea under the guidance of a tissue-specific promoter of the RuBP carboxylase/oxygenase small subunit (rbcS) gene. Regenerated transgenic plants with the cry1Ab expression cassette (cry1Ab-lox-bar-lox) showed the optimum insect motility rate (90%) in an in vitro insect bioassay with second instar larvae, signifying the insecticidal potency of Syn Cry1Ab. In parallel, another plant line was also generated with a chimaeric vector harbouring a cre recombinase gene under the control of the CaMV 2 × 35S promoter. Crossing between T1 plants with a single insertion of cry1Ab-lox-bar-lox T-DNA and T1 plants with moderate expression of a cre gene with a linked hygromycin resistance (hptII) gene was performed to exclude the bialaphos resistance (bar) marker gene. Excision of the bar gene was achieved in T1F1 hybrids, with up to 35.71% recombination frequency. Insect-resistant pigeon pea plants devoid of selectable marker genes (syn Cry1Ab- bar and cre-hptII) were established in a consecutive generation (T1F2) through genetic segregation.

205 ASSESSMENT OF DEVELOPMENTAL NEUROTOXICITY ON NEURAL DIFFERENTIATION IN HUMAN EMBRYONIC STEM CELLS

2014 ◽  
Vol 26 (1) ◽  
pp. 216
Author(s):  
H. S. Kang ◽  
E. M. Jung ◽  
E. B. Jeung
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Cytosine Arabinoside ◽  
Dopaminergic Neuron ◽  
Neural Differentiation ◽  
Marker Gene ◽  
Embryonic Stem ◽  
Penicillin G ◽  
Marker Genes

Human embryonic stem cells (hESCs) have a potential for differentiation into neuronal cells. Neural differentiation of hESCs is currently used in many fields of neurological study. Therefore, evaluation of developmental neurotoxicity in hESCs is possible during the embryonic period. In the current study, we investigated the neuronal toxicity of hydroxyurea, cytosine arabinoside, and penicillin G at multiple doses (low, medium, high) for 28 days for neural differentiation. For assessment of neural toxicity, we examined the expression of marker genes that represent neural cell development. The mRNA levels of the marker genes were evaluated by real-time PCR in hESCs. Morphological changes of hESCs during neuronal differentiation were also estimated. The results for low and medium doses of hydroxylurea showed a significant increase of dopaminergic neuron marker gene (NR4A2) and GABA neuron marker gene (GAD2). However, glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) showed a significant decrease. Results for another drug, cytosine arabinoside, showed a significant decrease of glutamartergic neuron marker gene (SLC1A2) and oligodendrocyte marker gene (CNP) at a high dose. In addition, cytosine arabinoside caused a significant decrease of dopaminergic neuron marker gene (NR4A2) without significant change in GAD2. For the control, penicillin G, no significant difference in expression of neural specific-genes was observed at all tested doses. These findings suggest that neural-specific genes are perturbed by hydroxyurea and cytosine arabinoside, which may be involved in abnormal neural development during the embryonic neurogenesis period in hESCs.

Sign in / Sign up

Export Citation Format

Share Document