scholarly journals Optimization of the TeraTox assay for preclinical teratogenicity assessment

2021 ◽  
Author(s):  
Manuela Jaklin ◽  
Jitao David Zhang ◽  
Nicole Schaefer ◽  
Nicole Clemann ◽  
Paul Barrow ◽  
...  
Keyword(s):  
Gene Expression ◽  
Machine Learning ◽  
Stem Cell ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies ◽  
Cell Renewal ◽  
Germ Layer

Teratogenicity poses severe threats to patient safety. Stem-cell-based in vitro systems are promising tools to predict human teratogenicity. However, current in vitro assays are limited because they either capture effects on a certain germ layer, or focus on a subset of predictive markers. Here we report the characterization and critical assessment of TeraTox, a newly developed multi-lineage differentiation assay using 3D human induced pluripotent stem cells. TeraTox probes stem-cell derived embryoid bodies with two endpoints, one quantifying cytotoxicity and the other inferring the teratogenicity potential with gene expression as a molecular phenotypic readout. To derive teratogenicity potentials from gene expression profiles, we applied both unsupervised machine-learning tools including factor analysis and supervised tools including classification and regression. To identify the best predictive model for the teratogenicity potential that is explainable, we systematically tested 64 machine-learning model architectures and identified the optimal model, which uses expression of 77 representative germ-layer genes, summarized by 10 latent germ-layer factors, as input for random-forest regression. We combined measured cytotoxicity and inferred teratogenicity potential to predict concentration-dependent teratogenicity profiles of 33 approved pharmaceuticals and 12 proprietary drug candidates with known in vivo data. Compared with the mouse embryonic stem cell test, which has been in routine use for more than a decade, the TeraTox assay shows higher sensitivity, particularly towards teratogens impairing ectodermal development or stem-cell renewal, and a more balanced prediction performance. We envision that further refinement and development of TeraTox has the potential to reduce and replace animal research in drug discovery and to improve preclinical assessment of teratogenicity.

scholarly journals Gene Expression Profiling of Functional Murine Embryonic Stem Cell-Derived Cardiomyocytes and Comparison with Adult Heart: Profiling of Murine ESC-Derived Cardiomyocytes

2009 ◽  
Vol 14 (3) ◽  
pp. 239-245 ◽  
Author(s):  
Tadahiro Shinozawa ◽  
Akiko Tsuji ◽  
Kenichi Imahashi ◽  
Kosuke Nakashima ◽  
Hiroshi Sawada ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Ion Channels ◽  
Embryonic Stem Cell ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Heart Tissue ◽  
Embryoid Bodies ◽  
Efficient Production ◽  
Adult Heart

Although embryonic stem cell (ESC)—derived cardiomyocytes may be a powerful tool in drug discovery, their potential has not yet been fully explored. Nor has a detailed comparison with adult heart tissue been performed. We have developed a method for efficient production of cardiomyocyte-rich embryoid bodies (EBs) from murine ESCs. Analysis of global gene expression profiles showed that EBs on day 7 and/or 21 of differentiation (d7CMs and d21CMs, respectively) were similar to adult heart tissue for genes categorized as regulators of muscle contraction or voltage-gated ion channel activity, although d21CMs were more mature than d7CMs for contractile components related to morphological structures. Calcium and sodium channel blockers altered Ca2+ transients, and isoproterenol, a β-adrenergic compound, increased the rate of beating in d7CMs and d21CMs. Our gene analytic system therefore enabled us to identify genes that are expressed in the physiological pathways associated with ion channels and structural components in d7CMs and d21CMs. We conclude that EBs might be of use for the basic screening of drugs that might affect contractile function through ion channels. ( Journal of Biomolecular Screening 2009:239-245)

scholarly journals Generation of functional heart organoids from mouse embryonic stem cells.

2020 ◽  
Author(s):  
Jiyoung Lee ◽  
Fumitoshi Ishino ◽  
Akito Sutani ◽  
Rin Kaneko
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Drug Testing ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies ◽  
Fibroblast Growth ◽  
Spatiotemporal Regulation

Abstract For the understanding of the spatiotemporal regulation of cardiogenesis, it is important to generate in vitro model of hearts. This protocol introduces how to generate heart organoids from mouse embryonic stem cell-derived embryoid bodies (EB) in the presence of the laminin-entactin (LN/ET) complex and fibroblast growth factor 4 (FGF4) by three consecutive steps, 1) the culture of ESCs, 2) in vitro differentiation of ESCs into EBs and 3) in vitro culture of EBs for the generation of heart organoids. The generated heart organoids possess structural and functional capacity similar to atrial and ventricular parts of in vivo embryonic heart. This simplified protocol also provides a promising research tool with a broad range of applications, including drug testing.

scholarly journals Effects of Natural Progesterone and Synthetic Progestin on Germ Layer Gene Expression in a Human Embryoid Body Model

2020 ◽  
Vol 21 (3) ◽  
pp. 769 ◽  
Author(s):  
Yoon Young Kim ◽  
Hoon Kim ◽  
Chang Suk Suh ◽  
Hung-Ching Liu ◽  
Zev Rosenwaks ◽  
...  
Keyword(s):  
Gene Expression ◽  
Embryonic Development ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Germ Layer ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Endometrial Cells ◽  
Natural Progesterone

Natural progesterone and synthetic progestin are widely used for the treatment of threatened abortion or in in vitro fertilization (IVF) cycles. This in vitro study aimed to assess whether the treatment with natural progesterone or synthetic progestin influences the germ layer gene expression on the early human embryonic development using human embryonic stem cells (hESCs)-derived embryoid bodies (hEBs) as a surrogate of early stage human embryonic development. Human EBs derived from hESCs were cultured for nine days, and were treated with natural progesterone (P4) or synthetic progestin, medroxyprogesterone acetate (MPA) at 10–7 M for five days. To reverse the effects of treatment, mifepristone (RU486) as progesterone antagonist was added to the hEBs for four days starting one day after the initiation of treatment. Mouse blastocysts (mBLs) were cultured in vitro for 24 h, and P4 or MPA at 10−7 M was treated for an additional 24 h. The treated embryos were further transferred onto in vitro cultured endometrial cells to evaluate chorionic gonadotropin (CG) expression. To analyze the effects of P4 or MPA, the expression of differentiation genes representing the three germ layers was investigated, GATA-binding factor 4 (GATA4), α-fetoprotein (AFP), hepatocyte nuclear factor (HNF)-3β, hepatocyte nuclear factor (HNF)-4α (endoderm), Brachyury, cardiac actin (cACT) (mesoderm), and Nestin (ectoderm), using quantitative reverse transcription PCR (qRT-PCR) and immunostaining. Significantly lower expressions of HNF-3β, HNF-4α, Brachyury, and Nestin were observed in MPA-treated hEBs (all p < 0.05), which was negated by RU486 treatment. This inhibitory effect of MPA was also observed in mouse embryos. Conclusively, the effects of natural progesterone and synthetic progestin may differ in the germ layer gene expression in the hEB model, which suggests that caution is necessary in the use of progestogen.

scholarly journals Identification of molecules derived from human fibroblast feeder cells that support the proliferation of human embryonic stem cells

2011 ◽  
Vol 16 (1) ◽  
Author(s):  
Sergey Anisimov ◽  
Nicolaj Christophersen ◽  
Ana Correia ◽  
Vanessa Hall ◽  
Ingrid Sandelin ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Molecular Mechanisms ◽  
Expression Profiles ◽  
Embryonic Stem ◽  
Feeder Cells ◽  
Continuous Growth

AbstractThe majority of human embryonic stem cell lines depend on a feeder cell layer for continuous growth in vitro, so that they can remain in an undifferentiated state. Limited knowledge is available concerning the molecular mechanisms that underlie the capacity of feeder cells to support both the proliferation and pluripotency of these cells. Importantly, feeder cells generally lose their capacity to support human embryonic stem cell proliferation in vitro following long-term culture. In this study, we performed large-scale gene expression profiles of human foreskin fibroblasts during early, intermediate and late passages using a custom DNA microarray platform (NeuroStem 2.0 Chip). The microarray data was validated using RT-PCR and virtual SAGE analysis. Our comparative gene expression study identified a limited number of molecular targets potentially involved in the ability of human neonatal foreskin fibroblasts to serve as feeder cells for human embryonic stem cell cultures. Among these, the C-KIT, leptin and pigment epithelium-derived factor (PEDF) genes were the most interesting candidates.

scholarly journals Shortening and Improving the Embryonic Stem Cell Test through the Use of Gene Biomarkers of Differentiation

2011 ◽  
Vol 2011 ◽  
pp. 1-8 ◽  
Author(s):  
Andrea C. Romero ◽  
Eugenio Vilanova ◽  
Miguel A. Sogorb
Keyword(s):  
Gene Expression ◽  
Risk Assessment ◽  
Stem Cell ◽  
Cell Viability ◽  
Embryonic Stem Cell ◽  
Total Duration ◽  
Embryonic Stem ◽  
End Points ◽  
Cell Test

The embryonic Stem cell Test (EST) is a validated assay for testing embryotoxicityin vitro. The total duration of this protocol is 10 days, and its main end-point is based on histological determinations. It is suggested that improvements on EST must be focused toward molecular end-points and, if possible, to reduce the total assay duration. Five days of exposure of D3 cells in monolayers under spontaneous differentiation to 50 ng/mL of the strong embryotoxic 5-fluorouracil or to 75 μg/mL of the weak embryotoxic 5,5-diphenylhydeantoin caused between 20 and 74% of reductions in the expression of the following genes:Pnpla6,Afp,Hdac7,Vegfa, andNes. The exposure to 1 mg/mL of nonembryotoxic saccharin only caused statistically significant reductions in the expression ofNes. These exposures reduced cell viability of D3 cells by 15, 28, and 34%. We applied these records to the mathematical discriminating function of the EST method to find that this approach is able to correctly predict the embryotoxicity of all three above-mentioned chemicals. Therefore, this work proposes the possibility of improve EST by reducing its total duration and by introducing gene expression as biomarker of differentiation, which might be very interesting forin vitrorisk assessment embryotoxicity.

scholarly journals Spontaneous induction of an homologous Robertsonian translocation, Rb(11.11) in a murine embryonic stem cell line

1990 ◽  
Vol 55 (2) ◽  
pp. 107-110 ◽  
Author(s):  
John Anthony Crolla ◽  
David Brown ◽  
David G. Whittingham
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Recombinant Dna ◽  
Genetic Manipulation ◽  
Embryonic Stem Cell Line ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Transgenic Animals ◽  
Translocation Chromosome

SummaryKaryotype analysis of a series of established mouse embryonic stem cell (MESC) lines showed that the majority were aneuploid by the 7th and 9th passage and that all lines contained a single Robertsonian (Rb) translocation chromosome with a symmetrical, homologous, arm composition Rb(11.11). Although the chromosomal imbalance makes these MESC lines unsuitable for genetic manipulation in vitro and hence for subsequent production of transgenic animals, the spontaneous occurrence and stable retention of the homologous Rb(11.11) as the only metacentric chromosome in an otherwise all acrocentric karyotype, provides potentially useful cell lines for gene assignment and recombinant DNA studies.

Simvastatin modulates mouse embryonic stem cell-derived chondrogenesis in vitro

2012 ◽  
Vol 26 (7) ◽  
pp. 1170-1176 ◽  
Author(s):  
J. Kramer ◽  
M. Bartsch ◽  
D. Krug ◽  
M. Klinger ◽  
M. Nitschke ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell

Cluster characterization of mouse embryonic stem cell-derived pluripotent embryoid bodies in four distinct developmental stages

Biologicals ◽  
2009 ◽  
Vol 37 (4) ◽  
pp. 235-244 ◽  
Author(s):  
J. Qin ◽  
X. Guo ◽  
G.H. Cui ◽  
Y.C. Zhou ◽  
D.R. Zhou ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Developmental Stages ◽  
Embryonic Stem ◽  
Mouse Embryonic Stem Cell ◽  
Embryoid Bodies
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