Evaluation of developmental toxicity using undifferentiated human embryonic stem cells

An embryonic stem cell test (EST) has been developed to evaluate the embryotoxic potential of chemicals with an in vitro system. In the present study, novel methods to screen toxic chemicals during the developmental process were evaluated using undifferentiated human embryonic stem (hES) cells. By using surface marker antigens (SSEA‐4, TRA‐1‐60 and TRA‐1‐81), we confirmed undifferentiated conditions of the used hES cells by immunocytochemistry. We assessed the developmental toxicity of embryotoxic chemicals, 5‐fluorouracil, indomethacin and non‐embryotoxic penicillin G in different concentrations for up to 7 days. While expressions of the surface markers were not significantly affected, the embryotoxic chemicals influenced their response to pluripotent ES cell markers, such as OCT‐4, NANOG, endothelin receptor type B (EDNRB), secreted frizzled related protein 2 (SFRP2), teratocarcinoma‐derived growth factor 1 (TDGF1), and phosphatase and tensin homolog (PTEN). Most of the pluripotent ES cell markers were down‐regulated in a dose‐dependent manner after treatment with embryotoxic chemicals. After treatment with 5‐fluorouracil, indomethacin and penicillin G, we observed a remarkable convergence in the degree of up‐regulation of development, cell cycle and apoptosis‐related genes by gene expression profiles using an Affymetrix GeneChips. Taken together, these results suggest that embryotoxic chemicals have cytotoxic effects, and modulate the expression of ES cell markers as well as development‐, cell cycle‐ and apoptosis‐related genes that have pivotal roles in undifferentiated hES cells. Therefore, we suggest that hES cells may be useful for testing the toxic effects of chemicals that could impact the embryonic developmental stage. Copyright © 2014 John Wiley & Sons, Ltd.     

Monitoring of gene expression in differentiation of embryoid bodies from cynomolgus monkey embryonic stem cells in the presence of bisphenol A

An embryonic stem (ES) cell differentiation model would facilitate analysis of developmental processes at the cellular level and the effects of embryotoxic and teratogenic factors in vitro. We explored the use of differentiation of embryoid bodies (EBs) from cynomolgus monkey ES cells for embryotoxicity testing. We determined the mRNA expression of various genes using real-time RT-PCR. Oct-3/4 expression was almost completely suppressed on day 14, suggesting that ES cells reached differentiated status in around 14 days. mRNA expression of E-cadherin, connexin 43, caveolin-1, and argininosuccinate synthetase was reproducibly suppressed during EB differentiation in 7-32% of ES cells in three separate experiments. Although these may not be "general stemness marker genes" such as Oct-3/4, they could play a role in readying stem cells for differentiation in response to deletion of signals from feeder cells. Next, we examined the effects of bisphenol A (BPA) on the mRNA expression of several differentiation marker genes for ES cells. That of PAX-6, an ectoderm marker, with 0, 0.1, and 10 microM BPA in 21-day EBs was 3,500%, 6,668%, and 8,394%, respectively, compared with ES cells. The difference between doses of 0 and 10 microM BPA in 21-day EBs was statistically significant (p=0.049). Pax-6 activation in the presence of BPA may interfere with the development of eyes, sensory organs, and certain neural and epidermal tissues usually derived from ectodermal tissues. Differentiation of EBs from cynomolgus monkey ES cells could be a useful model for detecting gene expression changes in response to chemical exposure.     

An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. II. Testing of compounds

The embryonic stem cell test (EST) developed by Spielmann et al. [Spielmann, H., Pohl, I., Doering, B., Liebsch, M., Moldenhauer, F., 1997. The embryonic stem cell test, an in vitro embryotoxicity test using two permanent mouse cell lines: 3T3 fibroblasts and embryonic stem cells. In Vitro. Toxicol. 10, 119–127] is currently the most promising in vitro assay to predict the embryotoxic potential of compounds. In this assay the disturbance of the differentiation of embryonic stem (ES) cells into contracting cardiomyocytes by test compounds as well as the direct cytotoxicity of the test compounds on ES cells and 3T3 fibroblasts is analyzed. On the basis of these results and by applying a biostatistical prediction model (PM) [Genschow, E., Scholz, G., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., Spielmann, H., 2000. Development of prediction models for three in vitro embryotoxicity tests in an ECVAM validation study. In Vitr. Mol. Toxicol. 13, 51–66; Genschow, E., Spielmann, H., Scholz, G., Pohl, I., Seiler, A., Clemann, N., Bremer, S., Becker, K., 2004. Validation of the embryonic stem cell test in the international ECVAM validation study on three in vitro embryotoxicity tests. Altern. Lab. Anim. 32, 209–244; Genschow, E., Spielmann, H., Scholz, G., Seiler, A., Brown, N., Piersma, A., Brady, M., Clemann, N., Huuskonen, H., Paillard, F., Bremer, S., Becker, K., 2002. The ECVAM international validation study on in vitro embryotoxicity tests: results of the definitive phase and evaluation of prediction models. European Centre for the Validation of Alternative Methods. Altern. Lab. Anim. 30, 151–176] test compounds can be classified as non-embryotoxic, weakly or strongly embryotoxic. In order to introduce a further endpoint into the EST, the disturbance of vasculogenesis and/or angiogenesis, a protocol to differentiate ES cells into endothelial cells, was established in the accompanying paper. PECAM-1 and VE-Cadherin gene expressions, quantified by real-time TaqMan^® PCR, were shown to be appropriate molecular markers for the differentiation of ES cells into endothelial cells. In the present study, the disturbance of the differentiation of ES cells into endothelial cells (i.e. the reduction in the expression of PECAM-1 and VE-Cadherin) by six test compounds with known embryotoxic potential was investigated: all-trans-retinoic acid (RA) and 5-fluorouracil (5-FU) are strongly embryotoxic, diphenylhydantoin (DPH) and valproic acid (Val) are weakly embryotoxic and saccharin (Sacch) and penicillin G (Pen G) are non-embryotoxic. In a first step the concentration of the test compound resulting in a 50% inhibition of PECAM-1 and VE-Cadherin gene expression and the concentration leading to a 50% decrease in the viability of ES cells and 3T3 fibroblasts were determined. In a second step and in a first attempt to assess the predictive potential of the newly developed test system the concentration values obtained were applied in the PM of the established EST to classify the selected test compounds. All six test compounds were correctly classified (i.e. the data obtained in vitro correlated with their known embryotoxic potential in vivo). Taken together it can be concluded that the disturbance of the differentiation of murine ES cells into endothelial cells represents a very promising new endpoint in a broadened EST with PECAM-1 and VE-Cadherin as specific differentiation marker genes.     

Simvastatin modulates mouse embryonic stem cell-derived chondrogenesis in vitro

It has been studied in detail that cellular differentiation during chondrogenesis can be recapitulated in vitro by differentiation of embryonic stem (ES) cells as embryoid bodies (EBs). We here used this model system of cartilage development to analyze the effect of simvastatin, a potentially embryotoxic substance. Statins are a group of drugs used to treat hypercholesterolaemia. We found that simvastatin activated cartilage nodule formation during EB differentiation. Extended application of simvastatin resulted in enhanced expression of cartilage marker molecules and prolonged persistence of cartilage nodules. Expression of collagen type II was upregulated during simvastatin-induced chondrogenic ES cell differentiation as demonstrated by quantitative real time PCR. However, immunostaining for cartilage marker molecules revealed that cartilage nodules within simvastatin-treated EBs were defective, bearing cavities of cell loss. Furthermore, caspase activity was reduced in comparison to untreated controls indicating reduced apoptosis. Taken together, we may speculate that simvastatin prolongs survival of chondrocytes and disrupts cellular integrity of cartilage nodules during EB development by affecting apoptotic mechanisms. The study underlines that ES cell-derived EBs are a useful in vitro model to screen substances for their embryotoxic and teratogenic potential.     

Differentiation of embryonic stem cells into hepatocytes that coexpress coagulation factors VIII and IX

Aim:

To establish an efficient culture system to support embryonic stem (ES) cell differentiation into hepatocytes that coexpress F-VIII and F-IX.

Methods:

Mouse E14 ES cells were cultured in differentiation medium containing sodium butyrate (SB), basic fibroblast growth factor (bFGF), and/or bone morphogenetic protein 4 (BMP4) to induce the differentiation of endoderm cells and hepatic progenitor cells. Hepatocyte growth factor, oncostatin M, and dexamethasone were then used to induce the maturation of ES cell–derived hepatocytes. The mRNA expression levels of endoderm-specific genes and hepatocyte-specific genes, including the levels of F-VIII and F-IX, were detected by RT-PCR and real-time PCR during various stages of differentiation. Protein expression was examined by immunofluorescence and Western blot. At the final stage of differentiation, flow cytometry was performed to determine the percentage of cells coexpressing F-VIII and F-IX, and ELISA was used to detect the levels of F-VIII and F-IX protein secreted into the culture medium.

Results:

The expression of endoderm-specific and hepatocyte-specific markers was upregulated to highest level in response to the combination of SB, bFGF, and BMP4. Treatment with the three inducers during hepatic progenitor differentiation significantly enhanced the mRNA and protein levels of F-VIII and F-IX in ES cell–derived hepatocytes. More importantly, F-VIII and F-IX were coexpressed with high efficiency at the final stage of differentiation, and they were also secreted into the culture medium.

Conclusion:

We have established a novel in vitro differentiation protocol for ES-derived hepatocytes that coexpress F-VIII and F-IX that may provide a foundation for stem cell replacement therapy for hemophilia.     

In vitro developmental toxicity test detects inhibition of stem cell differentiation by silica nanoparticles

While research into the potential toxic properties of nanomaterials is now increasing, the area of developmental toxicity has remained relatively uninvestigated. The embryonic stem cell test is an in vitro screening assay used to investigate the embryotoxic potential of chemicals by determining their ability to inhibit differentiation of embryonic stem cells into spontaneously contracting cardiomyocytes.Four well characterized silica nanoparticles of various sizes were used to investigate whether nanomaterials are capable of inhibition of differentiation in the embryonic stem cell test. Nanoparticle size distributions and dispersion characteristics were determined before and during incubation in the stem cell culture medium by means of transmission electron microscopy (TEM) and dynamic light scattering.Mouse embryonic stem cells were exposed to silica nanoparticles at concentrations ranging from 1 to 100 μg/ml. The embryonic stem cell test detected a concentration dependent inhibition of differentiation of stem cells into contracting cardiomyocytes by two silica nanoparticles of primary size 10 (TEM 11) and 30 (TEM 34) nm while two other particles of primary size 80 (TEM 34) and 400 (TEM 248) nm had no effect up to the highest concentration tested.Inhibition of differentiation of stem cells occurred below cytotoxic concentrations, indicating a specific effect of the particles on the differentiation of the embryonic stem cells. The impaired differentiation of stem cells by such widely used particles warrants further investigation into the potential of these nanoparticles to migrate into the uterus, placenta and embryo and their possible effects on embryogenesis.     

Analysis of altered gene expression specific to embryotoxic chemical treatment during embryonic stem cell differentiation into myocardiac and neural cells

Embryonic stem cells (ES cells), pluripotent cells derived from the inner cell mass of blastocysts, differentiate in vitro into a variety of cell types representing all three germ layers. They therefore constitute one of the most promising in vitro tools for developmental toxicology. To assess the developmental toxicity of chemicals using ES cells easily, identification of effective marker genes is a high priority. We report here altered gene expression during ES cell differentiation into myocardiac and neural cells on treatment with some embryotoxic and non-embryotoxic chemicals. Decreases in several undifferentiated markers such as Oct3/4 and Nanog, and elevated expression of genes associated with heart development or the central nervous system, respectively, were found on microarray analysis. Under differentiation of ES cells into myocardic cells, 107 genes were substantially up-regulated. Decrease in the expression of 13 genes of these (Hand1, Pim2, Tbx20, Myl4, Myl7, Hbb-bh1, Hba-a1, Col1a2, Hba-x, Cmya1, Pitx2, Smyd1 and Adam19) was observed specifically by embryotoxic chemicals. Of the 107 genes up-regulated under differentiation into neurons, 22 genes (Map2, Cpe, Marcks, Ptbp2, Sox11, Tubb2b, Vim, Arx, Emx2, Pax6, Basp1, Ddr1, Ndn, Sfrp, Ttc3, Ubqln2, Six3, Dcx, L1cam, Reln, Wnt1 and Nnat) showed reduced expression specifically by embryotoxic chemicals. Almost all gene sets identified in this study are known to be indispensable for differentiation and development of heart and brain tissues, and thus may serve in early detection or prediction of embryotoxicity of chemicals in vitro.     

Transferability of a modified embryonic stem cell test using a new endpoint for developmental neurotoxicity

We developed and analyzed a new surrogate endpoint of the mouse embryonic stem cell test (EST) for developmental neurotoxicity. To determine the sensitivity, specificity, and transferability of the new endpoint, a pre-validation team from three independent laboratories optimized and standardized the protocol for neuronal differentiation of mouse embryonic stem cells (mESCs) by measuring the neuronal differentiation rates of mESCs under different culture conditions, such as the presence or absence of basic fibroblast growth factor (bFGF) in the growth media and varying lengths of culture. In addition, a component ratio of neuronal cells was measured by using flow cytometry analysis of β-III tubulin (Tuj1)-positive cells and real-time polymerase chain reaction analysis of microtubule-associated protein 2 (MAP2) mRNA. Our results showed that the best growth was achieved by culturing mESCs for 12 d in N2B27 medium without bFGF or ascorbic acid. Lead (II) acetate and aroclor 1254 were used to test the usefulness of the new endpoint. When we used the known ID₅₀ values for lead (II) acetate in the EST model, it was classified as non-embryotoxic; however, when we used the new ID₅₀ values that we determined in this study, it was classified as weakly embryotoxic. Aroclor 1254 and penicillin G were also classified as weakly embryotoxic and non-embryotoxic compounds, respectively, when cardiac and neuronal differentiation ID₅₀ values were used. Therefore, our new surrogate endpoint for developmental neurotoxicity is not only sensitive and specific but also transferable among laboratories.     

The role of metabolism in the developmental toxicity of polycyclic aromatic hydrocarbon-containing extracts of petroleum substances

In vitro assays presently used for prenatal developmental toxicity (PDT) testing only assess the embryotoxic potential of parent substances and not that of potentially embryotoxic metabolites. Here we combined a biotransformation system, using hamster liver microsomes, with the ES-D3 cell differentiation assay of the embryonic stem cell test (EST) to compare the in vitro PDT potency of two 5-ring polycyclic aromatic hydrocarbons (PAHs), benzo[a]pyrene (BaP) and dibenz[a,h]anthracene (DBA), and dimethyl sulfoxide extracts from five PAH-containing petroleum substances (PS) and a gas-to-liquid base oil (GTLb), with and without bioactivation. In the absence of bioactivation, DBA, but not BaP, inhibited the differentiation of ES-D3 cells into beating cardiomyocytes in a concentration-dependent manner. Upon bioactivation, BaP induced in vitro PDT, while its major metabolite 3-hydroxybenzo[a]pyrene was shown to be active in the EST as well. This means BaP needs biotransformation to exert its embryotoxic effects. GTLb extracts tested negative in the EST, with and without bioactivation. The PS-induced PDT in the EST was not substantially changed following bioactivation, implying that metabolism may not play a crucial role for the PS extracts under study to exert the in vitro PDT effects. Altogether, these results indicate that although some PAH require bioactivation to induce PDT, some do not and this latter appears to hold for the (majority of) the PS constituents responsible for the in vitro PDT of these complex substances.     

Embryotoxicity hazard assessment of methylmercury and chromium using embryonic stem cells

The embryonic stem cell test (EST) has been scientifically validated (2001) as an in vitro embryotoxicity test, showing a good overall test accuracy of 78%. Methylmercury (MeHg) was the most significant outlayer identified, as the metal was the only strong in vivo embryotoxicant falsely predicted to be non-embryotoxic. The EST misclassification of MeHg, and the potential environmental exposure and developmental toxic hazards of heavy metals gave us the rationale to investigate whether the EST can correctly predict the embryotoxic potential of two heavy metals different from MeHg. The EST correctly classified trivalent chromium to be non-embryotoxic and hexavalent chromium to be embryotoxic, while we confirmed the misclassification of MeHg. MeHg causes developmental abnormalities in the brain. We therefore aimed to improve the in vitro prediction of MeHg embryotoxicity by including a neuronal ES cell differentiation assay. Differentiation of neuronal-like cells was demonstrated by real-time PCR experiments, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of nestin, neurofilament medium polypeptide, beta-tubulin III and microtubule-associated protein 2 (Mtap2) positive cells. We identified Mtap2 mRNA expression as a sensitive toxicological endpoint for MeHg-induced neuronal embryotoxicity, as Mtap2 mRNA was down-regulated in the presence of non-cytotoxic concentrations of MeHg. Noticeably, several other neuronal marker genes were unaffected by MeHg and Mtap2 expression was not affected until day 14 of differentiation. This implies that the total neuronal-like cell number was unchanged and that the down-regulation of Mtap2 expression reflects neuron-specific toxicity, i.e. instability of the neuron-specific microtubules, and arrest of the neuronal maturation. The fact, that most marker genes were unaffected by MeHg, stresses the importance of including an array of marker genes. In conclusion, our results imply that inclusion of additional target tissues and refinement of the current prediction model may enhance the predictive power of the EST.     

应用胚胎干细胞试验模型对碱性成纤维细胞生长因子发育毒性的初步评价

目的利用小鼠胚胎干细胞试验(EST)模型,初步评价bFGF的发育毒性。方法体外培养小鼠胚胎干细胞(ESC),通过形态学观察、碱性磷酸酶染色和RT-PCR方法进行ESC未分化鉴定;MTT法检测不同浓度bFGF对ESC和3T3细胞的毒性,RT-PCR半定量分析法检测bFGF对未分化基因Sox-2表达的影响。结果 bFGF对ESC和3T3的半数抑制浓度和ESC的半数抑制分化浓度分别为IC50ESC=15.2 mg.L-1,IC50 3T3=24.2 mg.L-1,ID50 ESC=1.7 mg.L-1。结论 bFGF发育毒性的判定为弱胚胎毒性。     

An in vitro embryotoxicity assay based on the disturbance of the differentiation of murine embryonic stem cells into endothelial cells. I: Establishment of the differentiation protocol

The aim of the present study was to establish an experimental protocol to differentiate murine embryonic stem (ES) cells into endothelial cells in vitro. The spinner flask technique as well as the hanging drop method were used to generate so-called embryoid bodies (EBs). In order to find out the optimal differentiation environment, EBs were cultured under various experimental conditions for up to 14 days. The influence of basic fibroblast growth factor (bFGF) alone, vascular endothelial growth factor (VEGF) alone, bFGF and VEGF together and a cocktail consisting of bFGF, VEGF, interleukin-6 (IL-6) and erythropoietin (Epo) on the induction of differentiation of ES cells into endothelial cells was studied. Different concentrations of growth factors and times of treatment were applied. Endothelial cells were characterized by analyzing the expression of platelet-endothelial cell adhesion molecule (PECAM-1), the endothelial-specific vascular endothelial cadherin (VE-Cadherin), the angiopoietin receptor Tie-2, VEGF receptors 1 and 2 (Flt-1 and Flk-1, respectively) and the soluble form of Flt-1 (sFlt) at the mRNA level. PECAM-1 and VE-Cadherin were also studied at the protein level. The data clearly showed that EBs generated by the hanging drop method, followed by their transfer into suspension culture on day 3 of differentiation and their subsequent plating on day 5 is the best of the studied methods to differentiate ES cells into endothelial cells. Addition of VEGF alone or a cocktail consisting of VEGF, bFGF, IL-6 and Epo resulted in the strongest gene expression levels of the above mentioned endothelial cell markers in the differentiated ES cells.     

An adverse outcome pathway framework for neural tube and axial defects mediated by modulation of retinoic acid homeostasis

Developmental toxicity can be caused through a multitude of mechanisms and can therefore not be captured through a single simple mechanistic paradigm. However, it may be possible to define a selected group of overarching mechanisms that might allow detection of the vast majority of developmental toxicants. Against this background, we have explored the usefulness of retinoic acid mediated regulation of neural tube and axial patterning as a general mechanism that, when perturbed, may result in manifestations of developmental toxicity that may cover a large part of malformations known to occur in experimental animals and in man. Through a literature survey, we have identified key genes in the regulation of retinoic acid homeostasis, as well as marker genes of neural tube and axial patterning, that may be used to detect developmental toxicants in in vitro systems. A retinoic acid–neural tube/axial patterning adverse outcome pathway (RA–NTA AOP) framework was designed. The framework was tested against existing data of flusilazole exposure in the rat whole embryo culture, the zebrafish embryotoxicity test, and the embryonic stem cell test. Flusilazole is known to interact with retinoic acid homeostasis, and induced common and unique NTA marker gene changes in the three test systems. Flusilazole-induced changes were similar in directionality to gene expression responses after retinoic acid exposure. It is suggested that the RA–NTA framework may provide a general tool to define mechanistic pathways and biomarkers of developmental toxicity that may be used in alternative in vitro assays for the detection of embryotoxic compounds.     

Embryonic stem cells as an in vitro model for mutagenicity, cytotoxicity and embryotoxicity studies: present state and future prospects

Primary cultures or established cell lines of vertebrates are commonly used to analyse the mutagenic, embryotoxic or teratogenic potential of environmental factors, drugs and xenobiotics in vitro. However, these cellular systems do not include developmental processes from early embryonic stages up to terminally differentiated cell types. An alternative approach has been offered by permanent lines of pluripotent stem cells of embryonic origin, such as embryonic carcinoma (EC), embryonic stem (ES) and embryonic germ (EG) cells. The undifferentiated stem cell lines are characterized by nearly unlimited self-renewal capacity and have been shown to differentiate in vitro into cells of all three primary germ layers. Pluripotent embryonic stem cell lines recapitulate cellular developmental processes and gene expression patterns of early embryogenesis during in vitro differentiation, data which are summarized in this review. In addition, recent studies are presented which investigated mutagenic, cytotoxic and embryotoxic effects of chemical substances using in vitro systems of pluripotent embryonic stem cells. Furthermore, an outlook is given on future molecular technologies using embryonic stem cells in developmental toxicology and embryotoxicology.     

Embryotoxicity assessment of developmental neurotoxicants using a neuronal endpoint in the embryonic stem cell test

The embryonic stem cell test (EST) is a validated in vitro embryotoxicity test; however, as the inhibition of cardiac differentiation alone is used as a differentiation endpoint in the EST, it may not be a useful test to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues. Previously, methylmercury (MeHg), cadmium and arsenic compounds, which are heavy metals that induce developmental neurotoxicity in vivo, were misclassified as nonembryotoxic with the EST. The aim of this study was to improve the EST to correctly screen such developmental neurotoxicants. We developed a neuronal endpoint (Tuj‐1 ID₅₀) using flow cytometry analysis of Tuj‐1‐positive cells to screen developmental neurotoxicants (MeHg, valproic acid, sodium arsenate and sodium arsenite) correctly using an adherent monoculture differentiation method. Using Tuj‐1 ID₅₀ in the EST instead of cardiac ID₅₀, all of the tested chemicals were classified as embryotoxic, while the negative controls were correctly classified as nonembryotoxic. To support the validity of Tuj‐1 ID₅₀, we compared the results from two experimenters who independently tested MeHg using our modified EST. An additional neuronal endpoint (MAP2 ID₅₀), obtained by analyzing the relative quantity of MAP2 mRNA, was used to classify the same chemicals. There were no significant differences in the three endpoint values of the two experimenters or in the classification results, except for isoniazid. In conclusion, our results indicate that Tuj‐1 ID₅₀ can be used as a surrogate endpoint of the traditional EST to screen developmental neurotoxicants correctly and it can also be applied to other chemicals. Copyright © 2011 John Wiley & Sons, Ltd.     

Hazard assessment of methylmercury toxicity to neuronal induction in embryogenesis using human embryonic stem cells

Pluripotent human embryonic stem cell (hESC) lines can to some extent mimic in vitro the development of the embryo, providing the scientific rationale for the use of these cells to establish tests for toxicity to embryogenesis. Such humanised in vitro tests have potential to improve human hazard prediction by avoiding interspecies differences. We explored the potential of a hESC-based assay for detection of toxicity to neuronal induction in embryonic development. Neuronal precursor differentiation was performed according to a previous publication, while we established a new protocol for maturation of the precursors into neuron-like cells. Appearance of neuronal derivatives was demonstrated by real-time PCR, showing up-regulation of several neuronal marker genes, and immunohistochemistry, demonstrating the appearance of neurofilament medium polypeptide, β-tubulin III and microtubule-associated protein 2 positive cells. In order to assess whether the hESC model could detect chemically induced developmental toxicity, we exposed the differentiating cells to methylmercury (MeHg) causing structural developmental abnormalities in the brain. Two separate exposure intervals were used to determine the effects of MeHg on neuronal precursor formation and their further maturation, respectively. The formation of precursors was sensitive to MeHg in non-cytotoxic concentrations, as the expression of several neuronal mRNA markers changed. In contrast, non-cytotoxic MeHg concentrations did not effect the mRNA marker expression in matured cells, indicating that neuronal precursor formation is more sensitive to MeHg than later stages of neuronal differentiation. Overall, our experiments demonstrate that the hESC assay can provide alerts for the adverse effects of MeHg on neuronal induction.     

Study of lead-induced neurotoxicity in neural cells differentiated from adipose tissue-derived stem cells

Abstract

In recent years, the use of stem cells as a new tool to create an in vitro model for toxicological studies has been considered. Adipose tissue-derived stem cells (ADSCs) are mesenchymal stem cells which have been extracted from adipose tissue by a less invasive method and rapidly propagated in culture medium compared with other sources. These cells have the capacity to differentiate into different cell lineage in vitro including neural cells. The aim of this study was to investigate the effect of lead exposure at various stages of differentiation on the neural differentiation of ADSCs. Third-passaged ADSCs were differentiated to neural cell in differentiation medium during 16?d. The ADSCs were exposed to lead (0.1–100?µg/ml) before differentiation and during differentiation on days 1, 7 and 14. The cell viability was assessed by MTT assay after 48?h. Also expression of β-tubulin III protein and Nestin, NeuN, NF70, Synaptophysin genes were evaluated at the end of differentiation in all treated groups. The results showed that lead had no effect on viability of undifferentiated ADSCs but differentiating cells showed various sensitivities to lead exposure and cells were more vulnerable to lead exposure at early stage of differentiation. Also, lead exposure at different stages of differentiation had various effects on gene expressions. Our study indicated that neural cells differentiated from ADSCs in vitro are sensitive to neurotoxic effect of lead as well-known developmental neurotoxicant, and then ADSCs could be a candidate as an alternative method for assessing neurodevelopmental toxicity potential of chemicals.