human-pluripotent-stem-cell-based-developmental-toxicity-assays-for

Human Pluripotent Stem Cell Based Developmental Toxicity Assays for Chemical Safety Screening and Systems Biology Data Generation

Efficient protocols to differentiate human pluripotent stem cells to various tissues in combination with -omics technologies opened up new horizons for in ...

University of Cologne

Cancer and cardiovascular disease (CVD) chemoprevention can be achieved by the use of natural, synthetic, or biologic compounds to reverse, suppress, or prevent the development of diseases. Chemoprevention is a potential anti-cancer approach, which has reduced secondary effects in comparison to classical prophylaxis. Natural compounds such as flavonoids reduce oxidative stress, which is the most likely mechanism in the protective effects of these compounds. Even though the exact mechanisms of action are not well understood another central action mechanism of polyphenolic flavonoids seems to be an induction of apoptosis as demonstrated in numerous cellular systems. Moreover, flavonoids may modulate protein and lipid kinase signaling pathways. Understanding the mechanism of these natural products will contribute to the development of more specific preventive strategies against cancer and CVD. Much of the research in the field is focused on epigallocatechin-3-O-gallate (EGCG), quercetin and curcumin, which were found to have beneficial effects against cancer and CVD. We review the chemoprotective mechanisms through which these natural compounds exert their beneficial effects against cancer and CVDs.

Chemoprotective mechanism of the natural compounds, epigallocatechin-3-O-gallate, quercetin and curcumin against cancer and cardiovascular diseases.

Human embryonic stem cells (hESCs) can be propagated indefinitely in vitro in an undifferentiated pluripotent state, can differentiate into derivatives of all three germ layers and are of considerable interest for applications in regenerative medicine. Clinical application of hESCs, however, requires reliable protocols for cryopreservation. Current protocols for cryopreservation of hESCs suffer from low recovery rates of hESCs and loss of pluripotency after thawing. We therefore studied the effects of cryopreservation on the viability, proliferation potential, and the pluripotency status of hESCs by combining cellular readouts and transcriptomics. We identified biological processes and pathways affected by cryopreservation in order to understand the limited survival rate of hESCs by comparing transcriptomes of hESCs at different time points after thawing with cells that did not undergo cryopreservation. While the transcriptomes of cells post thawing were very similar to those of control non-frozen hESCs for the early time points, we observed increased expression of genes involved in apoptosis, embryonic morphogenesis, ossification, tissue morphogenesis, regeneration, vasculature development and cell death at later time points. Our data suggest that inhibition of anoikis apoptosis and the stress-induced differentiation pathways are promising targets for improving the survival rate and maintaining pluripotency of hESCs after cryopreservation.

Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing.

Functional characterization and gene expression profiling of α-smooth muscle actin expressing cardiomyocytes derived from murine induced pluripotent stem cells.

The aim of this study was the development of an alternative testing method based on human embryonic stem cells for prenatal developmental toxicity with particular emphasis on early neural development. To this purpose, we designed an in vitro protocol based on the generation of neural rosettes, representing the in vitro counterpart of the developing neural plate and neural tube, and we challenged this complex cell model with retinoic acid (RA), a well-known teratogenic agent. The cells were exposed to different concentrations of RA during the process of rosettes formation. Morphological and molecular parameters were evaluated in treated as compared with untreated cells to detect both cytotoxicity and specific neural toxicity. Transcriptomic analysis was performed with microarray Affymetrix platform and validated by quantitative real-time PCR for genes relevant to early neural development such as HoxA1, HoxA3, HoxB1, HoxB4, FoxA2, FoxC1, Otx2, and Pax7. The results obtained demonstrated that neural rosette forming cells respond to RA with clear concentration-dependent morphological, and gene expression changes remarkably similar to those induced in vivo, in the developing neural tube, by RA exposure. This strict correspondence indicates that the neural rosette protocol described is capable of detecting specific teratogenic mechanisms causing perturbations of early neural development and therefore represents a promising alternative test for human prenatal developmental toxicity.

Development of a neural teratogenicity test based on human embryonic stem cells: response to retinoic acid exposure.

Chemopreventive agents are derived from edible plants and from ancient time is a part of daily intake for many humans and animals. There are several lines of compelling evidence from epidemiological, clinical and laboratory studies that these dietary constituents are associated in reducing cancer risks. However, developmental toxicity of these natural compounds cannot be excluded. In the present study, we examined the effect of chemopreventive agents on the differentiation of mouse embryonic stem cells (ESCs) as an in vitro embryotoxicity model. We assumed that inhibition of developmentally regulated genes in vitro might predict developmental toxicity also under in vivo conditions. We found that epigallocatechin gallate (EGCG) (20 microM) induced the expression of mesodermal and cardiomyocyte genes and a significant increase in the number and the percentage of cardiomyocytes. The increase of the subpopulation correlated with higher numbers of beating foci and beating frequencies. Curcumin on the other hand at 0.4 mM was seen to enhance expression of ectodermal transcripts. Quercetin (2.5 microM) was found to inhibit several developmentally regulated genes.

Effect of chemopreventive agents on differentiation of mouse embryonic stem cells.

Exposure to the antiepileptic drug valproic acid (VPA) during gestation causes neurofunctional and anatomic deficits in later life. At present, there are little human data on how early neural development is affected by chemicals. We used human embryonic stem cells, differentiating to neuroectodermal precursors, as a model to investigate the modes of action of VPA. Microarray expression profiling, qPCR of specific marker genes, immunostaining and the expression of green fluorescent protein under the control of the promoter of the canonical neural precursor cell marker HES5 were used as readouts. Exposure to VPA resulted in distorted marker gene expression, characterized by a relative increase in NANOG and OCT4 and a reduction in PAX6. A similar response pattern was observed with trichostatin A, a potent and specific histone deacetylase inhibitor (HDACi), but not with several other toxicants. Differentiation markers were disturbed by prolonged, but not by acute treatment with HDACi, and the strongest disturbance of differentiation was observed by toxicant exposure during early neural fate decision. The increased acetylation of histones observed in the presence of HDACi may explain the up-regulation of some genes. However, to understand the down-regulation of PAX6 and the overall complex transcript changes, we examined further epigenetic markers. Alterations in the methylation of lysines 4 and 27 of histone H3 were detected in the promoter region of PAX6 and OCT4. The changes in these activating and silencing histone marks provide a more general mechanistic rational for the regulation of developmentally important genes at non-cytotoxic drug concentrations.

Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome.

Embryonic development can be partially recapitulated in vitro by differentiating human embryonic stem cells (hESCs). Thalidomide is a developmental toxicant in vivo and acts in a species-dependent manner. Besides its therapeutic value, thalidomide also serves as a prototypical model to study teratogenecity. Although many in vivo and in vitro platforms have demonstrated its toxicity, only a few test systems accurately reflect human physiology. We used global gene expression and proteomics profiling (two dimensional electrophoresis (2DE) coupled with Tandem Mass spectrometry) to demonstrate hESC differentiation and thalidomide embryotoxicity/teratogenecity with clinically relevant dose(s). Proteome analysis showed loss of POU5F1 regulatory proteins PKM2 and RBM14 and an over expression of proteins involved in neuronal development (such as PAK2, PAFAH1B2 and PAFAH1B3) after 14 days of differentiation. The genomic and proteomic expression pattern demonstrated differential expression of limb, heart and embryonic development related transcription factors and biological processes. Moreover, this study uncovered novel possible mechanisms, such as the inhibition of RANBP1, that participate in the nucleocytoplasmic trafficking of proteins and inhibition of glutathione transferases (GSTA1, GSTA2), that protect the cell from secondary oxidative stress. As a proof of principle, we demonstrated that a combination of transcriptomics and proteomics, along with consistent differentiation of hESCs, enabled the detection of canonical and novel teratogenic intracellular mechanisms of thalidomide.

Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells.

Developmental neurotoxicity (DNT) and many forms of reproductive toxicity (RT) often manifest themselves in functional deficits that are not necessarily based on cell death, but rather on minor changes relating to cell differentiation or communication. The fields of DNT/RT would greatly benefit from in vitro tests that allow the identification of toxicant-induced changes of the cellular proteostasis, or of its underlying transcriptome network. Therefore, the 'human embryonic stem cell (hESC)-derived novel alternative test systems (ESNATS)' European commission research project established RT tests based on defined differentiation protocols of hESC and their progeny. Valproic acid (VPA) and methylmercury (MeHg) were used as positive control compounds to address the following fundamental questions: (1) Does transcriptome analysis allow discrimination of the two compounds? (2) How does analysis of enriched transcription factor binding sites (TFBS) and of individual probe sets (PS) distinguish between test systems? (3) Can batch effects be controlled? (4) How many DNA microarrays are needed? (5) Is the highest non-cytotoxic concentration optimal and relevant for the study of transcriptome changes? VPA triggered vast transcriptional changes, whereas MeHg altered fewer transcripts. To attenuate batch effects, analysis has been focused on the 500 PS with highest variability. The test systems differed significantly in their responses (<20 % overlap). Moreover, within one test system, little overlap between the PS changed by the two compounds has been observed. However, using TFBS enrichment, a relatively large 'common response' to VPA and MeHg could be distinguished from 'compound-specific' responses. In conclusion, the ESNATS assay battery allows classification of human DNT/RT toxicants on the basis of their transcriptome profiles.

Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach.

The present review assesses the current status of in vitro tests based on human pluripotent stem cell-derived toxicologically relevant target cells. The majority of the evaluated test systems are in the phase of test development. In particular the success rates of differentiation protocols and their reproducibility are varying depending on different culture conditions but also on the assessed marker panel and the functional evaluation of the cells. However, the amount of differentiated cells decreases in relation to their maturation status. No harmonization has been achieved yet about the required maturation status of the cellular models to be used for toxicological applications. Even with an established cellular model, the selection of appropriate readouts is challenging. Some areas of toxicity, such as developmental toxicity, suffer from insufficient knowledge on predictive biomarkers which leads to difficulties in the selection of the most appropriate endpoints. In this heterogeneous context the rapidly increasing knowledge about 'omics' technologies, might lead to an improvement of the current situation and allow the establishment of more predictive human in vitro toxicity tests.

Current status of human pluripotent stem cell based in vitro toxicity tests.

Human embryonic stem cells (hESCs) can be used to model the cellular and molecular mechanisms that underlie embryonic development. Understanding the cellular mechanisms and pathways involved in extraembryonic (ExE) differentiation is of great interest because of the important role of this process in maternal health and fertility. Fibroblast growth factor 2 (FGF-2) is widely used to maintain the self-renewal of hESCs and induced pluripotent stem cells, while all trans retinoic acid (RA) is used to facilitate the directed differentiation of hESCs. Here, we monitored the RA induced differentiation of hESCs to the ExE lineage with and without FGF-2 over a 7-day period via global transcriptional profiling. The stemness markers POU5F1, NANOG and TDGF1 were markedly downregulated, whereas an upregulation of the ExE markers KRT7, CGA, DDAH2 and IGFBP3 was observed. Many of the differentially expressed genes were involved in WNT and TGF-β signaling. RA inactivated WNT signaling even in the presence of exogenous FGF-2, which that promotes the maintenance of the pluripotent state. We also show that BMP4 was upregulated and that RA was able to modulate the TGF-β signaling pathway and direct hESCs toward the ExE lineage. In addition, an epigenetic study revealed hypermethylation of the DDAH2, TDGF1 and GATA3 gene promoters, suggesting a role for epigenetic regulation during ExE differentiation. These data reveals that the effect of RA prevails in the presence of exogenous FGF-2 thus resulting in the direction of hESCs toward the ExE lineage.

All-trans retinoic acid and basic fibroblast growth factor synergistically direct pluripotent human embryonic stem cells to extraembryonic lineages.

Pluripotent stem cells have great potential for regenerative medicine; however, their clinical use is associated with a risk of tumor formation. We utilized pluripotent cells expressing green fluorescent protein and puromycin resistance under control of the Oct4 promoter to study the persistence of potential pluripotent cells under embryoid body (EB) culture conditions, which are commonly used to obtain organotypic cells. We found that i.) OCT4-expressing cells dramatically decrease during the first week of differentiation, ii.) the number of OCT4-expressing cells recovers from day 7 on, iii.) the OCT4-expressing cells are similar to embryonic stem cells grown in the presence of leukemia inhibitory factor LIF but express several markers associated with germ cell formation, such as DAZL and STRA-8 and iv.) the persistence of potentially pluripotent cells is independent of supportive cells in EBs. Finally, OCT4-expressing cells, isolated from EBs after 2-month of culture, were further maintained under feeder-free conditions in absence of LIF and continued to express OCT4 in 95 % of the population for at least 36 days. These findings point to an alternative state of stable OCT4 expression. In the frame of the landscape model of differentiation two attractors of pluripotency might be defined based on their different characteristics.

Evidence for self-maintaining pluripotent murine stem cells in embryoid bodies.

A number of in vitro toxicity assays based on human embryonic stem cells (hESCs) are under development in order to provide alternative methods for the screening of chemicals and drugs and to reduce the number of animals needed for developmental toxicity assessment. The major challenge is to demonstrate the reliability of these in vitro methods by correlating the in vitro produced results to the available in vivo data. In this context transcriptomic approaches associated to toxicogenomic database analysis give the possibility to screen, annotate and cluster high numbers of genes and to identify the molecular changes that univocally mark the toxicity induced processes or are indicative of the early initiating events that lead to cellular toxicity. In this retrospective study we compare microarray transcriptomic data derived from two different hESCs lines (HUES1 and H9) exposed to valproic acid (VA) while applying the same differentiation protocol. We present the results of this comparative analysis in light of the known teratogenic effects of VA. The results show molecular changes in the processes of neural development, neural crest migration, apoptosis and regulation of transcription, indicating a good correspondence with the available in vivo data. We also describe common toxicological signatures and provide an interpretation of the observed qualitative differences referring to known biological features of the two hESCs lines.

Article	Year
Chemoprotective mechanism of the natural compounds, epigallocatechin-3-O-gallate, quercetin and curcumin against cancer and cardiovascular diseases. Current medicinal chemistry\| PubMed ID: 19355899	2009
Effects of cryopreservation on the transcriptome of human embryonic stem cells after thawing and culturing. Stem cell reviews\| PubMed ID: 21279480	2011
Functional characterization and gene expression profiling of α-smooth muscle actin expressing cardiomyocytes derived from murine induced pluripotent stem cells. Stem cell reviews\| PubMed ID: 21567216	2012
Development of a neural teratogenicity test based on human embryonic stem cells: response to retinoic acid exposure. Toxicological sciences : an official journal of the Society of Toxicology\| PubMed ID: 21934132	2011
Effect of chemopreventive agents on differentiation of mouse embryonic stem cells. Frontiers in bioscience (Elite edition)\| PubMed ID: 22201861	2012
Epigenetic changes and disturbed neural development in a human embryonic stem cell-based model relating to the fetal valproate syndrome. Human molecular genetics\| PubMed ID: 22723015	2012
Identification of thalidomide-specific transcriptomics and proteomics signatures during differentiation of human embryonic stem cells. PloS one\| PubMed ID: 22952932	2012
Human embryonic stem cell-derived test systems for developmental neurotoxicity: a transcriptomics approach. Archives of toxicology\| PubMed ID: 23179753	2013
Current status of human pluripotent stem cell based in vitro toxicity tests. Frontiers in bioscience (Scholar edition)\| PubMed ID: 23277040	2013
All-trans retinoic acid and basic fibroblast growth factor synergistically direct pluripotent human embryonic stem cells to extraembryonic lineages. Stem cell research\| PubMed ID: 23314291	2013
Evidence for self-maintaining pluripotent murine stem cells in embryoid bodies. Stem cell reviews\| PubMed ID: 24022811	2014
A comparative transcriptomic study on the effects of valproic acid on two different hESCs lines in a neural teratogenicity test system. Toxicology letters\| PubMed ID: 25192806	2014

Smita Jagtap

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