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 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.

Variant hepatocyte nuclear factor 1 is required for visceral endoderm specification

Development ◽  
1999 ◽  
Vol 126 (21) ◽  
pp. 4795-4805 ◽  
Author(s):  
E. Barbacci ◽  
M. Reber ◽  
M.O. Ott ◽  
C. Breillat ◽  
F. Huetz ◽  
...  
Keyword(s):  
Stem Cells ◽  
Embryonic Stem Cells ◽  
Embryonic Stem ◽  
Cell Lineage ◽  
Embryoid Bodies ◽  
Nuclear Factor ◽  
Visceral Endoderm ◽  
Hepatocyte Nuclear Factor ◽  
Parietal Endoderm ◽  
Nuclear Factor 1

Genetic and molecular evidence indicates that visceral endoderm, an extraembryonic cell lineage, is required for gastrulation, early anterior neural patterning, cell death and specification of posterior mesodermal cell fates. We show that variant Hepatocyte Nuclear Factor 1 (vHNF1), a homeodomain-containing transcription factor first expressed in the primitive endoderm, is required for the specification of visceral endoderm. vHnf1-deficient mouse embryos develop normally to the blastocyst stage, start implantation, but die soon afterwards, with abnormal or absent extraembryonic region, poorly organised ectoderm and no discernible visceral or parietal endoderm. However, immunostaining analysis of E5.5 nullizygous mutant embryos revealed the presence of parietal endoderm-like cells lying on an abnormal basal membrane. Homozygous mutant blastocyst outgrowths or differentiated embryonic stem cells do not express early or late visceral endoderm markers. In addition, in vHnf1 null embryoid bodies there is no activation of the transcription factors HNF-4alpha1, HNF1alpha and HNF-3gamma. Aggregation of vHnf1-deficient embryonic stem cells with wild-type tetraploid embryos, which contribute exclusively to extraembryonic tissues, rescues periimplantation lethality and allows development to progress to early organogenesis. Our results place vHNF1 in a preeminent position in the regulatory network that specifies the visceral endoderm and highlight the importance of this cell lineage for proper growth and differentiation of primitive ectoderm in pregastrulating embryos.

scholarly journals Evidence for Hepatocyte Differentiation from Embryonic Stem Cells In Vitro

2002 ◽  
Vol 11 (5) ◽  
pp. 429-434 ◽  
Author(s):  
Hitoshi Miyashita ◽  
Atsushi Suzuki ◽  
Katashi Fukao ◽  
Hiromitsu Nakauchi ◽  
Hideki Taniguchi
Keyword(s):  
Gene Expression ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Pcr Analysis ◽  
Liver Development ◽  
Hepatic Gene Expression ◽  
Hepatocyte Differentiation ◽  
Cell Based Therapy

We confirmed hepatocyte differentiation from embryonic stem (ES) cells in vitro. RT-PCR analysis revealed that a broad range of hepatic gene expression was observed in ES cells differentiated through formation of embryoid bodies (EBs) and its attachment culture. Quantitative PCR analysis revealed that hepatic gene expression related to early and late-stage liver development were enhanced through in vitro differentiation of ES cells. The presence of albumin-producing cells in the peripheral region of attached EBs was confirmed by immunocytochemical analysis. Future experiments will reveal the molecules that induce hepatocyte differentiation from ES cells in vitro. This research will provide systems for the investigation of mechanisms in liver development and establish a method of ES cell-based therapy for liver diseases.

scholarly journals Involvement of hepatocyte nuclear factor 3 in endoderm differentiation of embryonic stem cells.

1997 ◽  
Vol 17 (7) ◽  
pp. 3817-3822 ◽  
Author(s):  
M Levinson-Dushnik ◽  
N Benvenisty
Keyword(s):  
Phosphoenolpyruvate Carboxykinase ◽  
Es Cells ◽  
Embryonic Stem ◽  
Embryoid Bodies ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Endoderm Differentiation ◽  
Expression Of Genes ◽  
Alpha1 Antitrypsin

The transcription factors of the hepatocyte nuclear factor 3 (HNF3) family, which are active in the liver, are expressed early during endoderm differentiation. To study their involvement in early murine development, we examined their role in embryonic stem (ES) cells. HNF3alpha or HNF3beta mRNA transcripts were not detected in ES cells before differentiation, and only low levels of HNF3beta mRNA were detected at a late stage of differentiation of ES cells to embryoid bodies (EB) (20 days after induction of differentiation). To examine the consequences of overexpressing HNF3alpha or -beta in ES cells, we transfected the two genes into these cells and determined the levels of expression of tissue-specific genes during EB differentiation. Specifically, we examined expression of albumin, cystic fibrosis transmembrane conductance regulator (CFTR), phosphoenolpyruvate carboxykinase (PEPCK), alpha1-antitrypsin, transthyretin, zeta-globin, and neurofilament 68kd as markers for different cell lineages. Overexpression of HNF3beta (and to a lesser extent of HNF3alpha) induced the expression of genes associated with endodermal lineage, namely, the genes for CFTR and albumin, but did not induce the expression of genes involved in late endoderm differentiation, such as the genes for PEPCK and alpha1-antitrypsin. Moreover, expression of HNF1beta was highly induced in HNF3-overexpressing cells, while expression of HNF1alpha and HNF4 was only mildly induced in these cells. Therefore, HNF3alpha and -beta seem to be involved in early endoderm differentiation of ES cells and together with other developmental factors are apparently needed for the induction of the endodermal lineage in vivo.

scholarly journals Threshold Levels of Hepatocyte Nuclear Factor 6 (HNF-6) Acting in Synergy with HNF-4 and PGC-1α Are Required for Time-Specific Gene Expression during Liver Development

2006 ◽  
Vol 26 (16) ◽  
pp. 6037-6046 ◽  
Author(s):  
Jean-Bernard Beaudry ◽  
Christophe E. Pierreux ◽  
Graham P. Hayhurst ◽  
Nicolas Plumb-Rudewiez ◽  
Mary C. Weiss ◽  
...  
Keyword(s):  
Gene Expression ◽  
Specific Gene ◽  
Liver Development ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Loss Of Function ◽  
Specific Expression ◽  
Threshold Levels ◽  
Time Specific

ABSTRACT During liver development, hepatocytes undergo a maturation process that leads to the fully differentiated state. This relies at least in part on the coordinated action of liver-enriched transcription factors (LETFs), but little is known about the dynamics of this coordination. In this context we investigate here the role of the LETF hepatocyte nuclear factor 6 (HNF-6; also called Onecut-1) during hepatocyte differentiation. We show that HNF-6 knockout mouse fetuses have delayed expression of glucose-6-phosphatase (g6pc), which catalyzes the final step of gluconeogenesis and is a late marker of hepatocyte maturation. Using a combination of in vivo and in vitro gain- and loss-of-function approaches, we demonstrate that HNF-6 stimulates endogenous g6pc gene expression directly via a synergistic and interdependent action with HNF-4 and that it involves coordinate recruitment of the coactivator PGC-1α. The expression of HNF-6, HNF-4, and PGC-1α rises steadily during liver development and precedes that of g6pc. We provide evidence that threshold levels of HNF-6 are required to allow synergism between HNF-6, HNF-4, and PGC-1α to induce time-specific expression of g6pc. Our observations on the regulation of g6pc by HNF-6 provide a model whereby synergism, interdependency, and threshold concentrations of LETFs and coactivators determine time-specific expression of genes during liver development.

The responsiveness of embryonic stem cells to alpha and beta interferons provides the basis of an inducible expression system for analysis of developmental control genes

1993 ◽  
Vol 13 (12) ◽  
pp. 7971-7976
Author(s):  
L M Whyatt ◽  
A Düwel ◽  
A G Smith ◽  
P D Rathjen
Keyword(s):  
Gene Expression ◽  
Inner Cell Mass ◽  
Es Cells ◽  
Expression System ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Expression Vectors ◽  
Developmental Control ◽  
Developmental Potential

Embryonic stem (ES) cells, derived from the inner cell mass of the preimplantation mouse embryo, are used increasingly as an experimental tool for the investigation of early mammalian development. The differentiation of these cells in vitro can be used as an assay for factors that regulate early developmental decisions in the embryo, while the effects of altered gene expression during early embryogenesis can be analyzed in chimeric mice generated from modified ES cells. The experimental versatility of ES cells would be significantly increased by the development of systems which allow precise control of heterologous gene expression. In this paper, we report that ES cells are responsive to alpha and beta interferons (IFNs). This property has been exploited for the development of inducible ES cell expression vectors, using the promoter of the human IFN-inducible gene, 6-16. The properties of these vectors have been analyzed in both transiently and stably transfected ES cells. Expression was minimal or absent in unstimulated ES cells, could be stimulated up to 100-fold by treatment of the cells with IFN, and increased in linear fashion with increasing levels of IFN. High levels of induced expression were maintained for extended periods of time in the continuous presence of the inducing signal or following a 12-h pulse with IFN. Treatment of ES cells with IFN did not affect their growth or differentiation in vitro or compromise their developmental potential. This combination of features makes the 6-16-based expression vectors suitable for the functional analysis of developmental control control genes in ES cells.

Antagonism between apolipoprotein AI regulatory protein 1, Ear3/COUP-TF, and hepatocyte nuclear factor 4 modulates apolipoprotein CIII gene expression in liver and intestinal cells

1992 ◽  
Vol 12 (4) ◽  
pp. 1708-1718
Author(s):  
M Mietus-Snyder ◽  
F M Sladek ◽  
G S Ginsburg ◽  
C F Kuo ◽  
J A Ladias ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Protein ◽  
Lipid Binding ◽  
Hepatic Tissue ◽  
Nuclear Factor ◽  
Apolipoprotein Ai ◽  
Hepatocyte Nuclear Factor ◽  
Apolipoprotein Ciii ◽  
Caco2 Cells ◽  
Hepatocyte Nuclear Factor 4

Apolipoprotein CIII (apoCIII), a lipid-binding protein involved in the transport of triglycerides and cholesterol in the plasma, is synthesized primarily in the liver and the intestine. A cis-acting regulatory element, C3P, located at -90 to -66 upstream from the apoCIII gene transcriptional start site (+1), is necessary for maximal expression of the apoCIII gene in human hepatoma (HepG2) and intestinal carcinoma (Caco2) cells. This report shows that three members of the steroid receptor superfamily of transcription factors, hepatocyte nuclear factor 4 (HNF-4), apolipoprotein AI regulatory protein 1 (ARP-1), and Ear3/COUP-TF, act at the C3P site. HNF-4 activates apoCIII gene expression in HepG2 and Caco2 cells, while ARP-1 and Ear3/COUP-TF repress its expression in the same cells. HNF-4 activation is abolished by increasing amounts of ARP-1 or Ear3/COUP-TF, and repression by ARP-1 or Ear3/COUP-TF is alleviated by increasing amounts of HNF-4. HNF-4 and ARP-1 bind with similar affinities to the C3P site, suggesting that their opposing transcriptional effects may be mediated by direct competition for DNA binding. HNF-4 and ARP-1 mRNAs are present within the same cells in the liver and intestine, and protein extracts from hepatic tissue, HepG2, and Caco2 cells contain significantly more HNF-4 than ARP-1 or Ear3/COUP-TF binding activities. These findings suggest that the transcription of the apoCIII gene in vivo is dependent, at least in part, upon the intracellular balance of these positive and negative regulatory factors.

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